Monocyclic cyanoenones and methods of use thereof

ABSTRACT

The present invention features monocyclic cyanoenone compositions and methods for using the same in the treatment of diseases such as cancer, inflammatory diseases and neurodegenerative diseases.

The present application claims the benefit of priority to U.S.Provisional Application No. 61/082,550, filed Jul. 22, 2008, the entirecontents of this application being incorporated by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of biology andmedicine. More particularly, it concerns compounds and methods for thetreatment and prevention of diseases such as those associated withoxidative stress and inflammation.

II. Description of Related Art

The IκB kinases, IKKα and IKKβ, are related kinases that play a majorrole in the activation and regulation of the transcription factor,NF-κB. They are induced by stimuli such as TNFα and IL-1 tophosphorylate residue Ser³² and Ser³⁶ of IκBα, the regulatory subunit ofNF-κB. The IκB kinase (IKK) complex comprises three proteins: thecatalytic subunits, IKKα and IKKβ, and the regulatory subunit, IKKγ(NEMO). Without being bound by any particular mechanism or theory, thephosphorylation of IκBα results in ubiquitination and subsequentdegradation in the proteasome. This releases NF-κB dimers from thecytoplastic NF-κB-IκB complex, allowing NF-κB to translocate to thenucleus where it regulates the transcription of numerous target genes.IKKβ appears to be the principal kinase, whereas IKKα is not requiredfor activation of IKK and degradation of IκBα by proinflammatorystimuli. IKKβ triggers the activation of NF-κB in response to infectiousagents and proinflammatory cytokines, making it an attractive drugtarget for the treatment of inflammatory diseases. In addition, NF-κB isover-expressed or constitutively activated in many cancer cells where itinduces the expression of anti-apototic genes and/or suppression ofpro-apototic genes. A number of anticancer agents can also induce theactivation of NF-κB, which may culminate in the ability of the malignantcell to become drug resistant. Thus, the development of IKKβ inhibitorsrepresents potential therapeutics for the treatment of both cancer andinflammation (Karin et al., 2004).

The anti-inflammatory and anti-proliferative activity of the naturallyoccurring triterpenoid, oleanolic acid, has been improved by chemicalmodifications. For example,2-cyano-3,12-diooxooleana-1,9(11)-dien-28-oic acid (CDDO) and relatedcompounds have been developed (Honda et al., 1997; Honda et al., 1998;Honda et al., 1999; Honda et al., 2000; Honda et al., 2000; Honda, etal., 2002; Suh et al. 1998; Suh et al., 1999; Place et al., 2003; Libyet al., 2005). The methyl ester, CDDO-Me, is currently being evaluatedin phase II clinical trials for the treatment of melanoma, pancreaticcancer, diabetic nephropathy and chronic kidney disease.

Three-ringed compounds, whose rings A and C have enone functionalitiessimilar to those of CDDO, have been shown to be a novel class of potentanti-inflammatory, cytoprotective, growth suppressive, and proapoptoticcompounds (Favaloro et al., 2002; Honda et al., 2003; Honda et al.,2007). Among these compounds, TBE-31 was found to inhibit nitric oxide(NO) production at low nanomolecular concentrations in RAW cellsstimulated by interferon-γ (iNOS assay). Notably, orally active TBE-31is exceptionally potent against aflatoxin-induced liver cancer in rats(Liby et al., 2008). Furthermore, in vitro and in vivo potencies ofTBE-31 are much higher than those of CDDO.

Both CDDO and TBE-31 are multifunctional agents, which regulate proteinsinvolved in inflammation, oxidative stress, differentiation, apoptosis,and proliferation. Without being bound by any particular mechanism ortheory, these proteins, including, e.g., IKKβ, Keap1, and JAK1, areregulated by CDDO and TBE-31 by reversible and selective Michaeladdition between their cyanoenone functionality and the SH groups ofcysteine residues on these proteins (Scheme 1); Couch et al., 2005;Dinkova-Kostova et al., 2005). For example, Cys¹⁷⁹ on IKKβ wasidentified as one of targets of CDDO-Me (Ahmad et al., 2006). By bindingto this site, CDDO-Me blocks the binding of NF-κB to DNA and thusinhibits transcriptional activation. It has also been reported thatCDDO-Me inhibits the JAK1→STAT3 pathway by directly binding to JAK1 atCys¹⁰⁷⁷ and STAT3 at Cys²⁵⁹ (Ahmad et al., 2008). The small moleculeinhibitors of the STAT3 pathway are effective as anticancer agents invitro and in animal models.

Synthetic triterpenoid analogs of oleanolic acid have also been shown tobe inhibitors of cellular inflammatory processes, such as the inductionby IFN-γ of inducible nitric oxide synthase (iNOS) and of COX-2 in mousemacrophages. See Honda et al. (2000a); Honda et al. (2000b), and Hondaet al. (2002), which are all incorporated herein by reference. Forexample, one of these, 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acidmethyl ester (CDDO-Me), is currently in clinical trials for a variety ofdisorders related to inflammation, including cancer and diabeticnephropathy. Synthetic derivatives of another triterpenoid, betulinicacid, have also been shown to inhibit cellular inflammatory processes,although these compounds have been less extensively characterized (Hondaet al., 2006). The pharmacology of these synthetic triterpenoidmolecules is complex. Compounds derived from oleanolic acid have beenshown to affect the function of multiple protein targets and therebymodulate the activity of several important cellular signaling pathwaysrelated to oxidative stress, cell cycle control, and inflammation (e.g.,Dinkova-Kostova et al., 2005; Ahmad et al., 2006; Ahmad et al., 2008;Liby et al., 2007a). Derivatives of betulinic acid, though they haveshown comparable anti-inflammatory properties, also appear to havesignificant differences in their pharmacology compared to OA-derivedcompounds (Liby et al., 2007b). Further, it is not certain that thetriterpenoid starting materials employed to date have optimal propertiescompared to other possible starting materials. Given that the biologicalactivity profiles of known triterpenoid derivatives vary, and in view ofthe wide variety of diseases that may be treated or prevented withcompounds having potent antioxidant and anti-inflammatory effects, andthe high degree of unmet medical need represented within this variety ofdiseases, it is desirable to synthesize new compounds with diversestructures that may have improved biological activity profiles for thetreatment of one or more indications.

SUMMARY OF THE INVENTION

The present disclosure provides new monocyclic cyano enones (MCEs)antioxidant and anti-inflammatory properties, methods for theirmanufacture, and methods for their use.

In some aspects, the disclosure provides compounds of the formula:

wherein:

-   -   R₁, R₂, R₃, R₄, R₅ and R₆ are each independently:        -   hydrogen, hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18));    -   R₁ and R₃ are taken together and are alkanediyl_((C≦18)),        alkenediyl_((C≦18)), arenediyl_((C≦18)), alkoxydiyl_((C≦18)),        alkenyloxydiyl_((C≦18)), alkylaminodiyl_((C≦18)),        alkenylaminodiyl_((C≦18)), alkenylaminooxydiyl_((C≦18)),        alkenylaminothiodiyl_((C≦18)), with R₂, R₄, R₅ and R₆ as defined        above; or    -   R₃ and R₅ are taken together and are alkanediyl_((C≦18)),        alkenediyl_((C≦18)), arenediyl_((C≦18)), alkoxydiyl_((C≦18)),        alkenyloxydiyl_((C≦18)), alkylaminodiyl_((C≦18)),        alkenylaminodiyl_((C≦18)), alkenylaminooxydiyl_((C≦18)),        alkenylaminothiodiyl_((C≦18)), with R₁, R₂, R₄ and R₆ as defined        above;    -   provided that: R₄ is absent when the atom to which it is bound        forms part of a double bond; R₆ is absent when the atom to which        it is bound forms part of a double bond; neither R₁ nor R₂ is        hydrogen; and R₁ and R₂ are not both methyl;        or pharmaceutically acceptable salts, tautomers, prodrugs, or        optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₁ and R₂ are not            both methyl; and    -   R₇ and R₈ are each independently:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkylidene_((C≦6)),            alkoxy_((C≦6)), alkenyloxy_((C≦6)), alkynyloxy_((C≦6)),            aryloxy_((C≦6)), aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            alkylsulfonylamino_((C≦6)), amido_((C≦6)),            alkylimino_((C≦6)), alkenylimino_((C≦6)),            alkynylimino_((C≦6)), arylimino_((C≦6)),            aralkylimino_((C≦6)), heteroarylimino_((C≦6)),            heteroaralkylimino_((C≦6)), acylimino_((C≦6)),            alkylthio_((C≦6)), alkenylthio_((C≦6)), alkynylthio_((C≦6)),            arylthio_((C≦6)), aralkylthio_((C≦6)),            heteroarylthio_((C≦6)), heteroaralkylthio_((C≦6)),            acylthio_((C≦6)), thioacyl_((C≦6)), alkylsulfonyl_((C≦6)),            alkenylsulfonyl_((C≦6)), alkynylsulfonyl_((C≦6)),            arylsulfonyl_((C≦6)), aralkylsulfonyl_((C≦6)),            heteroarylsulfonyl_((C≦6)), heteroaralkylsulfonyl_((C≦6)),            alkylammonium_((C≦6)), alkylsulfonium_((C≦6)),            alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or a substituted            version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₁ and R₂ are not            both methyl; and    -   R₇ and R₈ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkoxy_((C≦6)),            alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),            aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            amido_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or            a substituted version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₁ and R₂ are not            both methyl; and    -   R₇ and R₈ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkoxy_((C≦6)),            alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),            aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            amido_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or            a substituted version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are each independently:        -   hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)),            heteroaryloxy_((C≦18)), aryloxy_((C≦18)), aralkoxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₁ and R₂ are not            both methyl; and    -   R₇ is:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkoxy_((C≦6)),            alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),            aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            amido_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or            a substituted version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₂ and R₅ are each independently:        -   hydrogen, hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₂ is not            hydrogen; and    -   R₇ and R₈ are each independently:        -   hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro,            imino, cyano, azido, mercapto, or thio; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkylidene_((C≦6)),            alkoxy_((C≦6)), alkenyloxy_((C≦6)), alkynyloxy_((C≦6)),            aryloxy_((C≦6)), aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            alkylsulfonylamino_((C≦6)), amido_((C≦6)),            alkylimino_((C≦6)), alkenylimino_((C≦6)),            alkynylimino_((C≦6)), arylimino_((C≦6)),            aralkylimino_((C≦6)), heteroarylimino_((C≦6)),            heteroaralkylimino_((C≦6)), acylimino_((C≦6)),            alkylthio_((C≦6)), alkenylthio_((C≦6)), alkynylthio_((C≦6)),            arylthio_((C≦6)), aralkylthio_((C≦6)),            heteroarylthio_((C≦6)), heteroaralkylthio_((C≦6)),            acylthio_((C≦6)), thioacyl_((C≦6)), alkylsulfonyl_((C≦6)),            alkenylsulfonyl_((C≦6)), alkynylsulfonyl_((C≦6)),            arylsulfonyl_((C≦6)), aralkylsulfonyl_((C≦6)),            heteroarylsulfonyl_((C≦6)), heteroaralkylsulfonyl_((C≦6)),            alkylammonium_((C≦6)), alkylsulfonium_((C≦6)),            alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or a substituted            version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₂ and R₅ are each independently:        -   hydrogen, hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₂ is not            hydrogen; and    -   R₇ and R₉ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkoxy_((C≦6)),            alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),            aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            amido_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or            a substituted version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₂ and R₅ are each independently:        -   hydrogen, hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), provided that R₂ is not            hydrogen; and    -   R₇ and R₉ are each independently:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, nitro, cyano,            azido, or mercapto; or        -   alkyl_((C≦6)), alkenyl_((C≦6)), alkynyl_((C≦6)),            aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),            heteroaralkyl_((C≦6)), acyl_((C≦6)), alkoxy_((C≦6)),            alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),            aralkoxy_((C≦6)), heteroaryloxy_((C≦6)),            heteroaralkoxy_((C≦6)), acyloxy_((C≦6)), alkylamino_((C≦6)),            dialkylamino_((C≦6)), alkoxyamino_((C≦6)),            alkenylamino_((C≦6)), alkynylamino_((C≦6)),            arylamino_((C≦6)), aralkylamino_((C≦6)),            heteroarylamino_((C≦6)), heteroaralkylamino_((C≦6)),            amido_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or            a substituted version of any of these groups;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some embodiments, the disclosure provides compounds of the formula:

wherein:

-   -   R₁ and R₂ are independently:        -   hydroxy, amino, cyano, or        -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),            aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),            heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),            alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),            aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),            heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),            alkylamino_((C≦18)), dialkylamino_((C≦18)),            alkoxyamino_((C≦18)), alkenylamino_((C≦18)),            alkynylamino_((C≦18)), arylamino_((C≦18)),            aralkylamino_((C≦18)), heteroarylamino_((C≦18)),            heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),            amido_((C≦18)), alkylideneamino_((C≦18)),            aralkylideneamino_((C≦18)), or a substituted version of any            of these groups, provided that R₁ and R₂ are not both            methyl;            or pharmaceutically acceptable salts, tautomers, prodrugs,            or optical isomers thereof.

In some variants of the above embodiments, the bond between atoms 4 and5 is a single bond. In other embodiments, the bond between atoms 4 and 5is a double bond.

In some variants of the above embodiments, R₂ is alkenyl_((C≦12)),substituted alkenyl_((C≦12)), alkynyl_((C≦12)) or substitutedalkynyl_((C≦12)). In other variants, R₂ is alkynyl_((C≦8)) orsubstituted alkynyl_((C≦8)). For example, R₂ can be —C≡C—R₉, wherein R₉is:

-   -   hydrogen or cyano; or    -   alkyl_((C≦6)), aryl_((C≦6)), acyl_((C≦6)), alkylsilyl_((C≦6)) or        a substituted version of any of these groups.

In variants of the above embodiments, R₉ is hydrogen. In other variants,R₉ is —Si(CH₃)₂C(CH₃)₃. In other variants, R₉ is cyano. In othervariants, R₉ is —CO₂H or —CO₂CH₃. R₉ is phenyl.

In some variants of the above embodiments, R₂ is alkenyl_((C≦8)) orsubstituted alkenyl_((C≦8)). For example, R₂ can be ethenyl (vinyl). Inother variants, R₂ is cyano. In other variants, R₂ is aralkyl_((C≦18)),substituted aralkyl_((C≦18)), heteroaralkyl_((C≦18)) or substitutedheteroaralkyl_((C≦18)). In other variants, R₂ is phenylmethyl. In othervariants, R₂ is alkyl_((C≦8)) or substituted alkyl_((C≦8)). For example,R₂ can be aminomethyl or hydroxymethyl. In other variants, R₂ isacyl_((C≦8)) or substituted acyl_((C≦8)). For example, R₂ can be —CO₂H,—C(O)NH₂ or —C(O)CH₃.

In some variants of the above embodiments, R₁ is alkyl_((C≦8)) orsubstituted alkyl_((C≦8)). For example, R₁ can be methyl, ethyl or—(CH₂)₂C(CH₃)₃. In other variants, R₁ is alkenyl_((C≦8)) or substitutedalkenyl_((C≦8)). For example, R₁ can be allyl. In other variants, R₁ isalkynyl_((C≦8)) or substituted alkynyl_((C≦8)). For example, R₁ can be—CH₂C≡CH. In other variants, R₁ is aralkyl_((C≦18)), substitutedaralkyl_((C≦18)), heteroaralkyl_((C≦18)) or substitutedheteroaralkyl_((C≦18)). For example, R₁ can be aralkyl_((C≦12)) orsubstituted aralkyl_((C≦12)). For example, R₁ can be phenylmethyl orphenylethyl.

In some variants of the above embodiments, R₁ is -L-R₁₀, wherein:

-   -   R₁₀ is:        -   hydrogen, hydroxy, halo, amino, hydroxyamino, cyano or            mercapto; or        -   alkyl_((C≦12)), alkenyl_((C≦12)), alkynyl_((C≦12)),            aryl_((C≦12)), aralkyl_((C≦12)), heteroaryl_((C≦12)),            heteroaralkyl_((C≦12)), acyl_((C≦12)), alkoxy_((C≦12)),            alkenyloxy_((C≦12)), alkynyloxy_((C≦12)), aryloxy_((C≦12)),            aralkoxy_((C≦12)), heteroaryloxy_((C≦12)),            heteroaralkoxy_((C≦12)), acyloxy_((C≦12)),            alkylamino_((C≦12)), dialkylamino_((C≦12)),            alkoxyamino_((C≦12)), alkenylamino_((C≦12)),            alkynylamino_((C≦12)), arylamino_((C≦12)),            aralkylamino_((C≦12)), heteroarylamino_((C≦12)),            heteroaralkylamino_((C≦12)), alkylsulfonylamino_((C≦12)),            amido_((C≦12)), alkylthio_((C≦12)), alkenylthio_((C≦12)),            alkynylthio_((C≦12)), arylthio_((C≦12)),            aralkylthio_((C≦12)), heteroarylthio_((C≦12)),            heteroaralkylthio_((C≦12)), acylthio_((C≦12)),            thioacyl_((C≦12)), alkylsulfonyl_((C≦12)),            alkenylsulfonyl_((C≦12)), alkynylsulfonyl_((C≦12)),            arylsulfonyl_((C≦12)), aralkylsulfonyl_((C≦12)),            heteroarylsulfonyl_((C≦12)), heteroaralkylsulfonyl_((C≦12)),            alkylammonium_((C≦12)), alkylsulfonium_((C≦12)),            alkylsilyl_((C≦12)), alkylsilyloxy_((C≦12)), or a            substituted version of any of these groups; and    -   L is alkanediyl_((C≦6)) or substituted alkanediyl_((C≦6)).

In some variants of the above embodiments, L is alkanediyl_((C1-3)). Forexample, L can be methanediyl, ethanediyl or propanediyl.

In some variants of the above embodiments, R₁₀ is alkyl_((C≦8)) or asubstituted version thereof. For example, R₁₀ can be tert-butyl. Inother variants, R₁₀ is aryl_((C≦12)), aralkyl_((C≦12)),heteroaryl_((C≦12)), heteroaralkyl_((C≦12)), or a substituted version ofany of these groups. For example, R₁₀ can be aryl_((C≦8)) or asubstituted version thereof. For example, R₁₀ can be phenyl. In othervariants, R₁₀ is amino, alkylamino_((C≦8)), substitutedalkylamino_((C≦8)), dialkylamino_((C≦8)) or substituteddialkylamino_((C≦8)). For example, R₁₀ can be amino. In other variants,R₁₀ is hydroxy, alkoxy_((C≦8)) or substituted alkoxy_((C≦8)). Forexample, R₁₀ can be hydroxy. In other variants, R₁₀ is acyl_((C≦8)) orsubstituted acyl_((C≦8)). For example, R₁₀ can be —CO₂H. In othervariants, R₁₀ is alkynyl_((C≦8)) or substituted alkynyl_((C≦8)). Forexample, R₁₀ can be —C≡C—R₁₁, wherein R₁₁ is:

-   -   hydrogen, hydroxy, halo, amino, hydroxyamino, cyano, mercapto,        or thio; or    -   alkyl_((C≦8)), alkenyl_((C≦8)), alkynyl_((C≦8)), aryl_((C≦8)),        aralkyl_((C≦8)), heteroaryl_((C≦8)), heteroaralkyl_((C≦8)),        acyl_((C≦8)), alkoxy_((C≦8)), alkenyloxy_((C≦8)),        alkynyloxy_((C≦8)), aryloxy_((C≦8)), aralkoxy_((C≦8)),        heteroaryloxy_((C≦8)), heteroaralkoxy_((C≦8)), acyloxy_((C≦8)),        alkylamino_((C≦8)), dialkylamino_((C≦8)), alkoxyamino_((C≦8)),        alkenylamino_((C≦8)), alkynylamino_((C≦8)), arylamino_((C≦8)),        aralkylamino_((C≦8)), heteroarylamino_((C≦8)),        heteroaralkylamino_((C≦8)), alkylsulfonylamino_((C≦8)),        amido_((C≦8)), alkylammonium_((C≦8)), alkylsulfonium_((C≦8)),        alkylsilyl_((C≦8)), alkylsilyloxy_((C≦8)), or a substituted        version of any of these groups.

In some variants of the above embodiments, R₁₁ is heteroaryl_((C≦8)),heteroaralkyl_((C≦8)), or substituted versions of either of thesegroups. For example, R₁₁ can be imidazoyl.

In some variants of the above embodiments, R₁ isaralkylideneamino_((C≦12)) or substituted aralkylideneamino_((C≦8)). Forexample, R₁ can be —N═CHCH₂Ph. In other variants, R₁ is cyano.

In some embodiments, the compound is further defined as:

wherein R₁ is:

-   -   cyano, or    -   alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),        aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),        heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),        alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),        aralkoxy_((C≦18)), heteroaryloxy_((C≦18)),        heteroaralkoxy_((C≦18)), acyloxy_((C≦18)), alkylamino_((C≦18)),        dialkylamino_((C≦18)), alkoxyamino_((C≦18)),        alkenylamino_((C≦18)), alkynylamino_((C≦18)),        arylamino_((C≦18)), aralkylamino_((C≦18)),        heteroarylamino_((C≦18)), heteroaralkylamino_((C≦18)),        alkylsulfonylamino_((C≦18)), amido_((C≦18)),        alkylideneamino_((C≦18)), aralkylideneamino_((C≦18)), or a        substituted version of any of these groups;        or pharmaceutically acceptable salts, tautomers, prodrugs, or        optical isomers thereof.

In some variants of the above embodiments, R₃ is hydrogen. In somevariants of the above embodiments, R₄ is hydrogen. In some variants ofthe above embodiments, R₅ is hydrogen. In some variants of the aboveembodiments, R₆ is hydrogen. In some variants of the above embodiments,R₇ is hydrogen. In some variants of the above embodiments, R_(g) ishydrogen.

Examples of compounds disclosed herein include:

In some embodiments, compounds of the present disclosure are in the formof pharmaceutically acceptable salts. In other embodiments, compounds ofthe present disclosure are not be in the form of a pharmaceuticallyacceptable salts.

In some embodiments, the compounds of the present disclosure are presentas mixtures of stereoisomers. In other embodiments, the compounds of thepresent disclosure are present as single stereoisomers.

In some embodiments, compounds of the present disclosure may be used asinhibitors of IFN-γ-induced nitrous oxide (NO) production inmacrophages, for example, having an IC₅₀ value of less than 0.2 μM.

Other general aspects of the present disclosure contemplate apharmaceutical composition comprising as an active ingredient a compoundof the present disclosure and a pharmaceutically acceptable carrier. Thecomposition may, for example, be adapted for administration by a routeselected from the group consisting of orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intrapleurally,intraprostaticaly, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,orally, parenterally, rectally, subconjunctival, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incrèmes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof. In particular embodiments, the compositionmay be formulated for oral delivery. In particular embodiments, thecomposition is formulated as a hard or soft capsule, a tablet, a syrup,a suspension, a wafer, or an elixir. In certain embodiments, the softcapsule is a gelatin capsule. Certain compositions may comprise aprotective coating, such as those compositions formulated for oraldelivery. Certain compositions further comprise an agent that delaysabsorption, such as those compositions formulated for oral delivery.Certain compositions may further comprise an agent that enhancessolubility or dispersibility, such as those compositions formulated fororal delivery. Certain compositions may comprise a compound of thepresent disclosure, wherein the compound is dispersed in a liposome, anoil and water emulsion or a water and oil emulsion.

Yet another general aspect of the present disclosure contemplates atherapeutic method comprising administering a pharmaceutically effectivecompound of the present disclosure to a subject. The subject may, forexample, be a human. These or any other methods of the presentdisclosure may further comprise identifying a subject in need oftreatment.

Another method of the present disclosure contemplates a method oftreating cancer in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure. The cancer may be any type of cancer, such as a carcinoma,sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma,or seminoma. Other types of cancers include cancer of the bladder,blood, bone, brain, breast, central nervous system, colon, endometrium,esophagus, genitourinary tract, head, larynx, liver, lung, neck, ovary,pancreas, prostate, spleen, small intestine, large intestine, stomach,or testicle. In these or any other methods, the subject may be aprimate. This or any other method may further comprise identifying asubject in need of treatment. The subject may have a family or patienthistory of cancer. In certain embodiments, the subject has symptoms ofcancer. The compounds disclosed herein may be administered via anymethod described herein, such as locally. In certain embodiments, thecompound is administered by direct intratumoral injection or byinjection into tumor vasculature. In certain embodiments, the compoundsmay be administered systemically. The compounds may be administeredintravenously, intra-arterially, intramuscularly, intraperitoneally,subcutaneously or orally, in certain embodiments.

In certain embodiments regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, thepharmaceutically effective amount is 0.1-1000 mg/kg. In certainembodiments, the pharmaceutically effective amount is administered in asingle dose per day. In certain embodiments, the pharmaceuticallyeffective amount is administered in two or more doses per day. Thecompound may be administered by contacting a tumor cell during ex vivopurging, for example. The method of treatment may comprise any one ormore of the following: a) inducing cytotoxicity in a tumor cell; b)killing a tumor cell; c) inducing apoptosis in a tumor cell; d) inducingdifferentiation in a tumor cell; or e) inhibiting growth in a tumorcell. The tumor cell may be any type of tumor cell, such as a leukemiacell. Other types of cells include, for example, a bladder cancer cell,a breast cancer cell, a lung cancer cell, a colon cancer cell, aprostate cancer cell, a liver cancer cell, a pancreatic cancer cell, astomach cancer cell, a testicular cancer cell, a brain cancer cell, anovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, abrain cancer cell, a bone cancer cell, or a soft tissue cancer cell.

Combination treatment therapy is also contemplated by the presentdisclosure. For example, regarding methods of treating cancer in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure, the method mayfurther comprise a treatment selected from the group consisting ofadministering a pharmaceutically effective amount of a second drug,radiotherapy, gene therapy, and surgery. Such methods may furthercomprise (1) contacting a tumor cell with the compound prior tocontacting the tumor cell with the second drug, (2) contacting a tumorcell with the second drug prior to contacting the tumor cell with thecompound, or (3) contacting a tumor cell with the compound and thesecond drug at the same time. The second drug may, in certainembodiments, be an antibiotic, anti-inflammatory, anti-neoplastic,anti-proliferative, anti-viral, immunomodulatory, or immunosuppressive.The second drug may be an alkylating agent, androgen receptor modulator,cytoskeletal disruptor, estrogen receptor modulator, histone-deacetylaseinhibitor, HMG-CoA reductase inhibitor, prenyl-protein transferaseinhibitor, retinoid receptor modulator, topoisomerase inhibitor, ortyrosine kinase inhibitor. In certain embodiments, the second drug is5-azacitidine, 5-fluorouracil, 9-cis-retinoic acid, actinomycin D,alitretinoin, all-trans-retinoic acid, annamycin, axitinib, belinostat,bevacizumab, bexarotene, bosutinib, busulfan, capecitabine, carboplatin,carmustine, CD437, cediranib, cetuximab, chlorambucil, cisplatin,cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunorubicin,decitabine, docetaxel, dolastatin-10, doxifluridine, doxorubicin,doxorubicin, epirubicin, erlotinib, etoposide, etoposide, gefitinib,gemcitabine, gemtuzumab ozogamicin, hexamethylmelamine, idarubicin,ifosfamide, imatinib, irinotecan, isotretinoin, ixabepilone, lapatinib,LBH589, lomustine, mechlorethamine, melphalan, mercaptopurine,methotrexate, mitomycin, mitoxantrone, MS-275, neratinib, nilotinib,nitrosourea, oxaliplatin, paclitaxel, plicamycin, procarbazine,semaxanib, semustine, sodium butyrate, sodium phenylacetate,streptozotocin, suberoylanilide hydroxamic acid, sunitinib, tamoxifen,teniposide, thiopeta, tioguanine, topotecan, TRAIL, trastuzumab,tretinoin, trichostatin A, valproic acid, valrubicin, vandetanib,vinblastine, vincristine, vindesine, or vinorelbine.

Methods of treating or preventing a disease with an inflammatorycomponent in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure are also contemplated. The disease may be, for example, lupusor rheumatoid arthritis. The disease may be an inflammatory boweldisease, such as Crohn's disease or ulcerative colitis. The disease withan inflammatory component may be a cardiovascular disease. The diseasewith an inflammatory component may be diabetes, such as type 1 or type 2diabetes. Compounds of the present disclosure may also be used to treatcomplications associated with diabetes. Such complications arewell-known in the art and include, for example, obesity, hypertension,atherosclerosis, coronary heart disease, stroke, peripheral vasculardisease, hypertension, nephropathy, neuropathy, myonecrosis, retinopathyand metabolic syndrome (syndrome X). The disease with an inflammatorycomponent may be a skin disease, such as psoriasis, acne, or atopicdermatitis. Administration of a compound of the present disclosure intreatment methods of such skin diseases may be, for example, topical ororal.

The disease with an inflammatory component may be metabolic syndrome(syndrome X). A patient having this syndrome is characterized as havingthree or more symptoms selected from the following group of fivesymptoms: (1) abdominal obesity; (2) hypertriglyceridemia; (3) lowhigh-density lipoprotein cholesterol (HDL); (4) high blood pressure; and(5) elevated fasting glucose, which may be in the range characteristicof Type 2 diabetes if the patient is also diabetic. Each of thesesymptoms is defined in the Third Report of the National CholesterolEducation Program Expert Panel on Detection, Evaluation and Treatment ofHigh Blood Cholesterol in Adults (Adult Treatment Panel III, or ATPIII), National Institutes of Health, 2001, NIH Publication No. 01-3670,incorporated herein by reference. Patients with metabolic syndrome,whether or not they have or develop overt diabetes mellitus, have anincreased risk of developing the macrovascular and microvascularcomplications that are listed above that occur with type 2 diabetes,such as atherosclerosis and coronary heart disease.

Another general method of the present disclosure entails a method oftreating or preventing a cardiovascular disease in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure. The cardiovascular disease may be,for example, atherosclerosis, cardiomyopathy, congenital heart disease,congestive heart failure, myocarditis, rheumatic heart disease, valvedisease, coronary artery disease, endocarditis, or myocardialinfarction. Combination therapy is also contemplated for such methods.For example, such methods may further comprise administering apharmaceutically effective amount of a second drug. The second drug maybe, for example, a cholesterol lowering drug, an anti-hyperlipidemic, acalcium channel blocker, an anti-hypertensive, or an HMG-CoA reductaseinhibitor. Non-limiting examples of second drugs include amlodipine,aspirin, ezetimibe, felodipine, lacidipine, lercanidipine, nicardipine,nifedipine, nimodipine, nisoldipine or nitrendipine. Other non-limitingexamples of second drugs include atenolol, bucindolol, carvedilol,clonidine, doxazosin, indoramin, labetalol, methyldopa, metoprolol,nadolol, oxprenolol, phenoxybenzamine, phentolamine, pindolol, prazosin,propranolol, terazosin, timolol or tolazoline. The second drug may be,for example, a statin, such as atorvastatin, cerivastatin, fluvastatin,lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin orsimvastatin.

Methods of treating or preventing a neurodegenerative disease in asubject, comprising administering to the subject a pharmaceuticallyeffective amount of a compound of the present disclosure are alsocontemplated. The neurodegenerative disease may, for example, beselected from the group consisting of Parkinson's disease, Alzheimer'sdisease, multiple sclerosis (MS), Huntington's disease and amyotrophiclateral sclerosis. In particular embodiments, the neurodegenerativedisease is Alzheimer's disease. In particular embodiments, theneurodegenerative disease is MS, such as primary progressive,relapsing-remitting secondary progressive or progressive relapsing MS.The subject may be, for example, a primate. The subject may be a human.

In particular embodiments of methods of treating or preventing aneurodegenerative disease in a subject, comprising administering to thesubject a pharmaceutically effective amount of a compound of the presentdisclosure, the treatment suppresses the demyelination of neurons in thesubject's brain or spinal cord. In certain embodiments, the treatmentsuppresses inflammatory demyelination. In certain embodiments, thetreatment suppresses the transection of neuron axons in the subject'sbrain or spinal cord. In certain embodiments, the treatment suppressesthe transection of neurites in the subject's brain or spinal cord. Incertain embodiments, the treatment suppresses neuronal apoptosis in thesubject's brain or spinal cord. In certain embodiments, the treatmentstimulates the remyelination of neuron axons in the subject's brain orspinal cord. In certain embodiments, the treatment restores lostfunction after an MS attack. In certain embodiments, the treatmentprevents a new MS attack. In certain embodiments, the treatment preventsa disability resulting from an MS attack.

One general aspect of the present disclosure contemplates a method oftreating or preventing a disorder characterized by overexpression ofiNOS genes in a subject, comprising administering to the subject apharmaceutically effective amount of a compound of the presentdisclosure.

Another general aspect of the present disclosure contemplates a methodof inhibiting IFN-γ-induced nitric oxide production in cells of asubject, comprising administering to said subject a pharmaceuticallyeffective amount of a compound of the present disclosure.

Yet another general method of the present disclosure contemplates amethod of treating or preventing a disorder characterized byoverexpression of COX-2 genes in a subject, comprising administering tothe subject a pharmaceutically effective amount of compound of thepresent disclosure.

Methods of treating renal/kidney disease (RKD) in a subject, comprisingadministering to the subject a pharmaceutically effective amount of acompound of the present disclosure are also contemplated. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. The RKD may result from, for example,a toxic insult. The toxic insult may result from, for example, animaging agent or a drug. The drug may be a chemotherapeutic, forexample. The RKD may result from ischemia/reperfusion injury, in certainembodiments. In certain embodiments, the RKD results from diabetes orhypertension. The RKD may result from an autoimmune disease. The RKD maybe further defined as chronic RKD, or acute RKD.

In certain methods of treating renal/kidney disease (RKD) in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure, the subject hasundergone or is undergoing dialysis. In certain embodiments, the subjecthas undergone or is a candidate to undergo kidney transplant. Thesubject may be a primate. The primate may be a human. The subject inthis or any other method may be, for example, a cow, horse, dog, cat,pig, mouse, rat or guinea pig.

Also contemplated by the present disclosure is a method for improvingglomerular filtration rate or creatinine clearance in a subject,comprising administering to the subject a pharmaceutically effectiveamount of a compound of the present disclosure.

Kits are also contemplated by the present disclosure, such as a kitcomprising: a compound of the present disclosure; and instructions whichcomprise one or more forms of information selected from the groupconsisting of indicating a disease state for which the compound is to beadministered, storage information for the compound, dosing informationand instructions regarding how to administer the compound. The kit maycomprise a compound of the present disclosure in a multiple dose form.

In certain embodiments, compounds of the present disclosure may be usedin preventing and treating diseases and disorders whose pathologyinvolves oxidative stress, inflammation, and dysregulation ofinflammatory signaling pathways. In particular embodiments, compoundsdisclosed herein may be used in treating diseases and disorderscharacterized by overexpression of inducible nitric oxide synthase(iNOS), inducible cyclooxygenase (COX-2), or both, in affected tissues;high levels of production of reactive oxygen species (ROS) or reactivenitrogen species (RNS) such as superoxide, hydrogen peroxide, nitricoxide or peroxynitrite; or excessive production of inflammatorycytokines or other inflammation-related proteins such as TNFα, IL-6,IL-1, IL-8, ICAM-1, VCAM-1, and VEGF. Such diseases or disorders may, insome embodiments, involve undesirable proliferation of certain cells, asin the case of cancer (e.g., solid tumors, leukemias, myelomas,lymphomas, and other cancers), fibrosis associated with organ failure,or excessive scarring. Other such disorders include (but are not limitedto) autoimmune diseases such as lupus, rheumatoid arthritis,juvenile-onset diabetes, multiple sclerosis, psoriasis, and Crohn'sdisease; cardiovascular diseases such as atherosclerosis, heart failure,myocardial infarction, acute coronary syndrome, restenosis followingvascular surgery, hypertension, and vasculitis; neurodegenerative orneuromuscular diseases such as Alzheimer's disease, Parkinson's disease,Huntington's disease, ALS, and muscular dystrophy; neurologicaldisorders such as epilepsy and dystonia; neuropsychiatric conditionssuch as major depression, bipolar disorder, post-traumatic stressdisorder, schizophrenia, anorexia nervosa, ADHD, and autism-spectrumdisorders; retinal diseases such as macular degeneration, diabeticretinopathy, glaucoma, and retinitis; chronic and acute pain syndromes,including inflammatory and neuropathic pain; hearing loss and tinnitus;diabetes and complications of diabetes, including metabolic syndrome,diabetic nephropathy, diabetic neuropathy, and diabetic ulcers;respiratory diseases such as asthma, chronic obstructive pulmonarydisease, acute respiratory distress syndrome, and cystic fibrosis;inflammatory bowel diseases; osteoporosis, osteoarthritis, and otherdegenerative conditions of bone and cartilage; acute or chronic organfailure, including renal failure, liver failure (including cirrhosis andhepatitis), and pancreatitis; ischemia-reperfusion injury associatedwith thrombotic or hemorrhagic stroke, subarachnoid hemorrhage, cerebralvasospasm, myocardial infarction, shock, or trauma; complications oforgan or tissue transplantation including acute or chronic transplantfailure or rejection and graft-versus-host disease; skin diseasesincluding atopic dermatitis and acne; sepsis and septic shock; excessiveinflammation associated with infection, including respiratoryinflammation associated with influenza and upper respiratory infections;mucositis associated with cancer therapy, including radiation therapy orchemotherapy; and severe burns.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The invention may be better understood by reference to oneof these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-D show how the UV spectra of four MCEs change in the presenceof dithiothreitol (DTT) (MCE-1, FIG. 1A; MCE-2, FIG. 1B; MCE-3, FIG. 1C)or NaOH (MCE-4 (dhMCE-1), FIG. 1D).

FIG. 2 shows the UV spectra of MCE-1 in different solvents.

FIG. 3 shows the UV spectra of MCE-1 in different solvents. In contrastto FIG. 2, the lower absorbance range is expanded.

FIG. 4 shows how the UV spectrum of MCE-5 changes in the presence ofdithiothreitol (DTT).

FIG. 5 shows how the presence of base effects the UV spectra of thedhMCE-5 tautomers.

FIG. 6 shows how the UV spectrum of MCE-10 changes in the presence ofdithiothreitol (DTT).

FIG. 7 shows how the UV spectrum of MCE-10 changes in the presence ofdithiothreitol (DTT). In contrast to FIG. 6, the lower absorbance rangeis expanded.

FIG. 8 shows how the UV spectrum of MCE-12 changes in the presence ofdithiothreitol (DTT).

FIG. 9 shows how the UV spectrum of MCE-12 changes in the presence ofdithiothreitol (DTT). In contrast to FIG. 8, the lower absorbance rangeis expanded.

FIG. 10 shows how the UV spectrum of MCE-15 changes in the presence ofdithiothreitol (DTT). The solid line corresponds to the UV spectrum inthe absence of DTT, the dashed line corresponds to the UV spectrum inthe presence of 0.1 mM DTT, and the dashed-and-dotted line correspondsto the UV spectrum in the presence of 1 mM DTT.

FIG. 11 shows how the UV spectrum of MCE-7 changes in the presence ofdithiothreitol (DTT). The solid line corresponds to the UV spectrum inthe absence of DTT, the dashed line corresponds to the UV spectrum inthe presence of 0.1 mM DTT, and the dashed-and-dotted line correspondsto the UV spectrum in the presence of 1 mM DTT.

FIG. 12 shows how the UV spectrum of MCE-13 changes in the presence ofdithiothreitol (DTT). The solid line corresponds to the UV spectrum inthe absence of DTT, the dashed line corresponds to the UV spectrum inthe presence of 0.1 mM DTT, and the dashed-and-dotted line correspondsto the UV spectrum in the presence of 1 mM DTT.

FIG. 13 shows that MCE-1 gives a reversible Michael adduct by ¹H-NMR. Inthe ¹H NMR (500 MHz, DMSO-d₆ at 21° C.) of MCE-1, the olefinic protonsH_(A), H_(B), and H_(C) are observed at δ 8.17 ppm (d, J=3 Hz), 7.21 ppm(dd, J=3 and 10 Hz), and 6.35 ppm (d, J=10 Hz), respectively, whileolefinic protons H_(A), H_(B), and H_(C) of MCE-1 decrease, an enolproton [at δ 11.12 ppm (brs)] and new olefinic protons H_(D) and H_(E)appear and increase according to increasing amounts of DTT.

FIG. 14 shows how the ¹H-NMR spectrum of MCE-1+1 eq. of DTT (500 MHz,DMSO-d₆ at 21° C.) changes as the temperature is increased.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Definitions

When used in the context of a chemical group, “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “halo” means independently —F, —Cl,—Br or —I; “amino” means —NH₂ (see below for definitions of groupscontaining the term amino, e.g., alkylamino); “hydroxyamino” means—NHOH; “nitro” means —NO₂; imino means ═NH (see below for definitions ofgroups containing the term imino, e.g., alkylimino); “cyano” means —CN;“azido” means —N₃; in a monovalent context “phosphate” means —OP(O)(OH)₂or a deprotonated form thereof; in a divalent context “phosphate” means—OP(O)(OH)O— or a deprotonated form thereof; “mercapto” means —SH;“thio” means ═S; “thioether” means —S—; “sulfonamido” means —NHS(O)₂—(see below for definitions of groups containing the term sulfonamido,e.g., alkylsulfonamido); “sulfonyl” means —S(O)₂—(see below fordefinitions of groups containing the term sulfonyl, e.g.,alkylsulfonyl); “sulfinyl” means —S(O)—(see below for definitions ofgroups containing the term sulfinyl, e.g., alkylsulfinyl); and “silyl”means —SiH₃ (see below for definitions of group(s) containing the termsilyl, e.g., alkylsilyl).

The symbol “—” means a single bond, “=” means a double bond, and “≡”means triple bond. The symbol

represents a single bond or a double bond. The symbol

, when drawn perpendicularly across a bond indicates a point ofattachment of the group. It is noted that the point of attachment istypically only identified in this manner for larger groups in order toassist the reader in rapidly and unambiguously identifying a point ofattachment. The symbol

means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol

means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol

means a single bond where the conformation is unknown (e.g., either R orS), the geometry is unknown (e.g., either E or Z) or the compound ispresent as mixture of conformation or geometries (e.g., a 50%/50%mixture).

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed.

When a group “R” is depicted as a “floating group” on a fused ringsystem, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fuzed rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

When y is 2 and “(R)_(y)” is depicted as a floating group on a ringsystem having one or more ring atoms having two replaceable hydrogens,e.g., a saturated ring carbon, as for example in the formula:

then each of the two R groups can reside on the same or a different ringatom. For example, when R is methyl and both R groups are attached tothe same ring atom, a geminal dimethyl group results. Where specificallyprovided for, two R groups may be taken together to form a divalentgroup, such as one of the divalent groups further defined below. Whensuch a divalent group is attached to the same ring atom, a spirocyclicring structure will result.

In the case of a double-bonded R group (e.g., oxo, imino, thio,alkylidene, etc.), any pair of implicit or explicit hydrogen atomsattached to one ring atom can be replaced by the R group. This conceptis exemplified below:

For the groups below, the following parenthetical subscripts furtherdefine the groups as follows: “(Cn)” defines the exact number (n) ofcarbon atoms in the group. “(Cn)” defines the maximum number (n) ofcarbon atoms that can be in the group, with the minimum number of carbonatoms in such at least one, but otherwise as small as possible for thegroup in question. E.g., it is understood that the minimum number ofcarbon atoms in the group “alkenyl_((C≦8))” is two. For example,“alkoxy_((C≦10))” designates those alkoxy groups having from 1 to 10carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms). (Cn-n′) defines both theminimum (n) and maximum number (n′) of carbon atoms in the group.Similarly, “alkyl_((C2-10))” designates those alkyl groups having from 2to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any rangederivable therein (e.g., 3 to 10 carbon atoms)).

The term “alkyl” when used without the “substituted” modifier refers toa non-aromatic monovalent group with a saturated carbon atom as thepoint of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, no carbon-carbon double or triple bonds, and no atoms otherthan carbon and hydrogen. The groups, —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr), —CH(CH₃)₂ (iso-Pr), —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl), —CH₂CH(CH₃)₂(iso-butyl), —C(CH₃)₃ (tert-butyl), —CH₂C(CH₃)₃ (neo-pentyl),cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl arenon-limiting examples of alkyl groups. The term “substituted alkyl”refers to a non-aromatic monovalent group with a saturated carbon atomas the point of attachment, a linear or branched, cyclo, cyclic oracyclic structure, no carbon-carbon double or triple bonds, and at leastone atom independently selected from the group consisting of N, O, F,Cl, Br, I, Si, P, and S. The following groups are non-limiting examplesof substituted alkyl groups: —CH₂OH, —CH₂Cl, —CH₂Br, —CH₂SH, —CF₃,—CH₂CN, —CH₂C(O)H, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)NHCH₃,—CH₂C(O)CH₃, —CH₂OCH₃, —CH₂OCH₂CF₃, —CH₂OC(O)CH₃, —CH₂NH₂, —CH₂NHCH₃,—CH₂N(CH₃)₂, —CH₂CH₂Cl, —CH₂CH₂OH, —CH₂CF₃, —CH₂CH₂OC(O)CH₃,—CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The term “alkanediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkanediyl group isattached with two σ-bonds, The groups, —CH₂— (methylene), —CH₂CH₂—,—CH₂C(CH₃)₂CH₂—, —CH₂CH₂CH₂—, and

are non-limiting examples of alkanediyl groups. The term “substitutedalkanediyl” refers to a non-aromatic monovalent group, wherein thealkanediyl group is attached with two σ-bonds, with one or two saturatedcarbon atom(s) as the point(s) of attachment, a linear or branched,cyclo, cyclic or acyclic structure, no carbon-carbon double or triplebonds, and at least one atom independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. The following groups arenon-limiting examples of substituted alkanediyl groups: —CH(F)—, —CF₂—,—CH(Cl)—, —CH(OH)—, —CH(OCH₃)—, and —CH₂CH(Cl)—.

The term “alkenyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples of alkenyl groups include: —CH═CH₂ (vinyl), —CH═CHCH₃,—CH═CHCH₂CH₃, —CH₂CH═CH₂ (allyl), —CH₂CH═CHCH₃, and —CH═CH—C₆H₅. Theterm “substituted alkenyl” refers to a monovalent group with anonaromatic carbon atom as the point of attachment, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds, alinear or branched, cyclo, cyclic or acyclic structure, and at least oneatom independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, arenon-limiting examples of substituted alkenyl groups.

The term “alkenediyl” when used without the “substituted” modifierrefers to a divalent group that is nonaromatic prior to attachment,wherein the alkenediyl group is attached with two σ-bonds, which maybecome aromatic upon attachment, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. The groups, —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, —CH═CH—CH═CH—, and

are non-limiting examples of alkenediyl groups. The term “substitutedalkenediyl” refers to a divalent group that is nonaromatic prior toattachment, wherein the alkenediyl group is attached with two σ-bonds,which may become aromatic upon attachment, with two carbon atoms aspoints of attachment, a linear or branched, cyclo, cyclic or acyclicstructure, at least one nonaromatic carbon-carbon double bond, nocarbon-carbon triple bonds, and at least one atom independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Thefollowing groups are non-limiting examples of substituted alkenediylgroups: —CF═CH—, —C(OH)═CH—, and —CH₂CH═C(Cl)—.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent group with a nonaromatic carbon atom as the point ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡CH, —C≡CCH₃, —C≡CC₆H₅ and —CH₂C≡CCH₃, arenon-limiting examples of alkynyl groups. The term “substituted alkynyl”refers to a monovalent group with a nonaromatic carbon atom as the pointof attachment and at least one carbon-carbon triple bond, a linear orbranched, cyclo, cyclic or acyclic structure, and at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. The group, —C≡CSi(CH₃)₃, is a non-limiting example of asubstituted alkynyl group.

The term “alkynediyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and no atoms other than carbon andhydrogen. The groups, —C≡C—, —C≡CCH₂—, and —C≡CCH(CH₃)— are non-limitingexamples of alkynediyl groups. The term “substituted alkynediyl” refersto a non-aromatic divalent group, wherein the alkynediyl group isattached with two σ-bonds, with two carbon atoms as points ofattachment, a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon triple bond, and at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups —C≡CCFH— and —C≡CHCH(Cl)— are non-limiting examples ofsubstituted alkynediyl groups.

The term “aryl” when used without the “substituted” modifier refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of one or more six-memberedaromatic ring structure(s) wherein the ring atoms are all carbon, andwherein the monovalent group consists of no atoms other than carbon andhydrogen. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl),—C₆H₄CH₂CH₂CH₃ (propylphenyl), —C₆H₄CH(CH₃)₂, C₆H₄CH(CH₂)₂,—C₆H₃(CH₃)CH₂CH₃ (methylethylphenyl), —C₆H₄CH—CH₂ (vinylphenyl),—C₆H₄CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and the monovalentgroup derived from biphenyl. The term “substituted aryl” refers to amonovalent group with an aromatic carbon atom as the point ofattachment, said carbon atom forming part of one or more six-memberedaromatic ring structure(s) wherein the ring atoms are all carbon, andwherein the monovalent group further has at least one atom independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S,Non-limiting examples of substituted aryl groups include the groups:—C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄I, —C₆H₄OH, —C₆H₄OCH₃, —C₆H₄OCH₂CH₃,—C₆H₄OC(O)CH₃, —C₆H₄NH₂, —C₆H₄NHCH₃, —C₆H₄N(CH₃)₂, —C₆H₄CHO,—C₆H₄CH₂OC(O)CH₃, —C₆H₄CH₂NH₂, —C₆H₄CF₃, —C₆H₄CN, —C₆H₄CHO, —C₆H₄CHO,—C₆H₄C(O)CH₃, —C₆H₄C(O)C₆H₅, —C₆H₄CO₂H, —C₆H₄CO₂CH₃, —C₆H₄CONH₂,—C₆H₄CONHCH₃, and —C₆H₄CON(CH₃)₂.

The term “arenediyl” when used without the “substituted” modifier refersto a divalent group, wherein the arenediyl group is attached with twoσ-bonds, with two aromatic carbon atoms as points of attachment, saidcarbon atoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen.Non-limiting examples of arenediyl groups include:

The term “substituted arenediyl” refers to a divalent group, wherein thearenediyl group is attached with two σ-bonds, with two aromatic carbonatoms as points of attachment, said carbon atoms forming part of one ormore six-membered aromatic rings structure(s), wherein the ring atomsare carbon, and wherein the divalent group further has at least one atomindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples of aralkyls are: phenylmethyl(benzyl, Bn), 1-phenyl-ethyl, 2-phenyl-ethyl, indenyl and2,3-dihydro-indenyl, provided that indenyl and 2,3-dihydro-indenyl areonly examples of aralkyl in so far as the point of attachment in eachcase is one of the saturated carbon atoms. When the term “aralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the aryl is substituted. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, 2-oxo-2-phenyl-ethyl(phenylcarbonylmethyl), 2-chloro-2-phenyl-ethyl, chromanyl where thepoint of attachment is one of the saturated carbon atoms, andtetrahydroquinolinyl where the point of attachment is one of thesaturated atoms.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent group with an aromatic carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of an aromatic ring structure wherein at least one of thering atoms is nitrogen, oxygen or sulfur, and wherein the monovalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofaryl groups include acridinyl, furanyl, imidazoimidazolyl,imidazopyrazolyl, imidazopyridinyl, imidazopyrimidinyl, indolyl,indazolinyl, methylpyridyl, oxazolyl, phenylimidazolyl, pyridyl,pyrrolyl, pyrimidyl, pyrazinyl, quinolyl, quinazolyl, quinoxalinyl,tetrahydroquinolinyl, thienyl, triazinyl, pyrrolopyridinyl,pyrrolopyrimidinyl, pyrrolopyrazinyl, pyrrolotriazinyl,pyrroloimidazolyl, chromenyl (where the point of attachment is one ofthe aromatic atoms), and chromanyl (where the point of attachment is oneof the aromatic atoms). The term “substituted heteroaryl” refers to amonovalent group with an aromatic carbon atom or nitrogen atom as thepoint of attachment, said carbon atom or nitrogen atom forming part ofan aromatic ring structure wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the monovalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroarenediyl” when used without the “substituted” modifierrefers to a divalent group, wherein the heteroarenediyl group isattached with two σ-bonds, with an aromatic carbon atom or nitrogen atomas the point of attachment, said carbon atom or nitrogen atom formingpart of one or more aromatic ring structure(s) wherein at least one ofthe ring atoms is nitrogen, oxygen or sulfur, and wherein the divalentgroup consists of no atoms other than carbon, hydrogen, aromaticnitrogen, aromatic oxygen and aromatic sulfur. Non-limiting examples ofheteroarenediyl groups include:

The term “substituted heteroarenediyl” refers to a divalent group,wherein the heteroarenediyl group is attached with two σ-bonds, with anaromatic carbon atom or nitrogen atom as points of attachment, saidcarbon atom or nitrogen atom forming part of one or more six-memberedaromatic ring structure(s), wherein at least one of the ring atoms isnitrogen, oxygen or sulfur, and wherein the divalent group further hasat least one atom independently selected from the group consisting ofnon-aromatic nitrogen, non-aromatic oxygen, non aromatic sulfur F, Cl,Br, I, Si, and P.

The term “heteroaralkyl” when used without the “substituted” modifierrefers to the monovalent group -alkanediyl-heteroaryl, in which theterms alkanediyl and heteroaryl are each used in a manner consistentwith the definitions provided above. Non-limiting examples of aralkylsare: pyridylmethyl, and thienylmethyl. When the term “heteroaralkyl” isused with the “substituted” modifier, either one or both the alkanediyland the heteroaryl is substituted.

The term “acyl” when used without the “substituted” modifier refers to amonovalent group with a carbon atom of a carbonyl group as the point ofattachment, further having a linear or branched, cyclo, cyclic oracyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the oxygen atom of the carbonyl group. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)C₆H₄CH₂CH₃,—COC₆H₃(CH₃)₂, and —C(O)CH₂C₆H₅, are non-limiting examples of acylgroups. The term “acyl” therefore encompasses, but is not limited togroups sometimes referred to as “alkyl carbonyl” and “aryl carbonyl”groups. The term “substituted acyl” refers to a monovalent group with acarbon atom of a carbonyl group as the point of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, furtherhaving at least one atom, in addition to the oxygen of the carbonylgroup, independently selected from the group consisting of N, O, F, Cl,Br, I, Si, P, and S. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl), —CO₂CH₃(methylcarboxyl), CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃, —CO₂C₆H₅, CO₂CH(CH₃)₂,CO₂CH(CH₂)₂, —C(O)NH₂ (carbamoyl), —C(O)NHCH₃, —C(O)NHCH₂CH₃,—CONHCH(CH₃)₂, —CONHCH(CH₂)₂, —CON(CH₃)₂, —CONHCH₂CF₃, —CO-pyridyl,—CO-imidazoyl, and —C(O)N₃, are non-limiting examples of substitutedacyl groups. The term “substituted acyl” encompasses, but is not limitedto, “heteroaryl carbonyl” groups.

The term “alkylidene” when used without the “substituted” modifierrefers to the divalent group ═CRR′, wherein the alkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, alkenyl, or R and R′ are taken togetherto represent alkanediyl or alkenediyl. Non-limiting examples ofalkylidene groups include: ═CH₂, ═CH(CH₂CH₃), and ═C(CH₃)₂. The term“substituted alkylidene” refers to the group ═CRR′, wherein thealkylidene group is attached with one σ-bond and one π-bond, in which Rand R′ are independently hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, provided that either one of R and R′ is asubstituted alkyl or substituted alkenyl, or R and R′ are taken togetherto represent a substituted alkanediyl or a substituted alkenediyl.

The term “aralkylidene” when used without the “substituted” modifierrefers to the divalent group ═CRR′, wherein the aralkylidene group isattached with one σ-bond and one π-bond, in which R and R′ areindependently hydrogen, alkyl, aryl or aralkyl, provided that at leastone of R and R′ is aryl or aralkyl. The term “substituted aralkylidene”refers to the group ═CRR′, wherein the aralkylidene group is attachedwith one σ-bond and one π-bond, in which R and R′ are independentlyhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl orsubstituted aralkyl, provided that at least one of R and R′ is aryl,substituted aryl, aralkyl or substituted aralkyl.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkoxy groups include: —OCH₃, —OCH₂CH₃,—OCH₂CH₂CH₃, —OCH(CH₃)₂, —OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl.The term “substituted alkoxy” refers to the group —OR, in which R is asubstituted alkyl, as that term is defined above. For example, —OCH₂CF₃is a substituted alkoxy group.

The term “alkoxydiyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkoxydiylgroup isattached with two σ-bonds, with (a) two saturated carbon atoms as pointsof attachment, (b) one saturated carbon atom and one oxygen atom aspoints of attachment, or (c) two oxygen atoms as points of attachment,further having a linear or branched, cyclo, cyclic or acyclic structure,no carbon-carbon double or triple bonds in the group's backbone, furtherhaving no backbone atoms other than carbon or oxygen and having at leastone of each of these atoms in the group's backbone, and no side chainscomprising groups other than hydrogen or alkyl. The groups, —O—CH₂CH₂—,—CH₂—O—CH₂CH₂—, —O—CH₂CH₂—O— and —O—CH₂—O— are non-limiting examples ofalkoxydiyl groups. The term “substituted alkanyloxydiyl” refers to adivalent group that is attached with two σ-bonds, with (a) two saturatedcarbon atoms as points of attachment, (b) one saturated carbon atom andone oxygen atom as points of attachment, or (c) two oxygen atoms aspoints of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, no carbon-carbon double or triple bonds, and atleast one atom independently selected from the group consisting of N, F,Cl, Br, I, Si, P, and S, or having additional oxygen atoms beyond thosein the group's backbone. The following groups are non-limiting exampleof a substituted alkoxydiylgroups: —O—CH₂C(OH)H—O— and —O—CH₂C(Cl)H—O—.

Similarly, the terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”,“heteroaryloxy”, “heteroaralkoxy” and “acyloxy”, when used without the“substituted” modifier, refers to groups, defined as —OR, in which R isalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively, as those terms are defined above. When any of the termsalkenyloxy, alkynyloxy, aryloxy, aralkyloxy and acyloxy is modified by“substituted,” it refers to the group —OR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “alkenyloxydiyl” when used without the “substituted” modifierrefers to a divalent group that is nonaromatic prior to attachment,wherein the alkenyloxydiyl group is attached with two σ-bonds, which maybecome aromatic upon attachment, with (a) two carbon atoms as points ofattachment, (b) one carbon atom and one oxygen atom as points ofattachment, or (c) two oxygen atoms as points of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon double bond that is non-aromatic at least priorto attachment, further having no backbone atoms other than carbon oroxygen and having at least one of each of these atoms in the group'sbackbone, and no side chains comprising groups other than hydrogen oralkyl. The groups, —O—CH═CH—, —O—CH═CHO— and —O—CH═CHCH₂— arenon-limiting examples of alkenyloxydiyl groups. The term “substitutedalkenyloxydiyl” refers to a divalent group that is nonaromatic prior toattachment, wherein the substituted alkenyloxydiyl group is attachedwith two σ-bonds, which may become aromatic upon attachment, with (a)two carbon atoms as points of attachment, (b) one carbon atom and oneoxygen atom as points of attachment, or (c) two oxygen atoms as pointsof attachment, further having a linear or branched, cyclo, cyclic oracyclic structure, at least one carbon-carbon double bond that isnon-aromatic at least prior to attachment and at least one atomindependently selected from the group consisting of N, F, Cl, Br, I, Si,P, and S, or having additional oxygen atoms beyond those in the group'sbackbone. The following groups are non-limiting example of a substitutedalkenyloxydiyl groups: —O—CH═C(OH)—O— and —O—CH═C(Cl)—O—.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylamino groups include:—NHCH₃, —NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH(CH₂)₂,—NHCH₂CH₂CH₂CH₃, —NHCH(CH₃)CH₂CH₃, —NHCH₂CH(CH₃)₂, —NHC(CH₃)₃,—NH-cyclopentyl, and —NH-cyclohexyl. The term “substituted alkylamino”refers to the group —NHR, in which R is a substituted alkyl, as thatterm is defined above. For example, —NHCH₂CF₃ is a substitutedalkylamino group.

The term “dialkylamino” when used without the “substituted” modifierrefers to the group —NRR′, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl having two or more saturated carbon atoms, at least two ofwhich are attached to the nitrogen atom. Non-limiting examples ofdialkylamino groups include: —NHC(CH₃)₃, —N(CH₃)CH₂CH₃, —N(CH₂CH₃)₂,N-pyrrolidinyl, and N-piperidinyl. The term “substituted dialkylamino”refers to the group —NRR′, in which R and R′ can be the same ordifferent substituted alkyl groups, one of R or R′ is an alkyl and theother is a substituted alkyl, or R and R′ can be taken together torepresent a substituted alkanediyl with two or more saturated carbonatoms, at least two of which are attached to the nitrogen atom.

The terms “alkoxyamino”, “alkenylamino”, “alkynylamino”, “arylamino”,“aralkylamino”, “heteroarylamino”, “heteroaralkylamino”, and“alkylsulfonylamino” when used without the “substituted” modifier,refers to groups, defined as —NHR, in which R is alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively, as those terms are defined above. A non-limiting exampleof an arylamino group is —NHC₆H₅. When any of the terms alkoxyamino,alkenylamino, alkynylamino, arylamino, aralkylamino, hetero aryl amino,heteroaralkylamino and alkylsulfonylamino is modified by “substituted,”it refers to the group —NHR, in which R is substituted alkoxy, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and alkylsulfonyl,respectively.

The term “amido” (acylamino), when used without the “substituted”modifier, refers to the group —NHR, in which R is acyl, as that term isdefined above. A non-limiting example of an acylamino group is—NHC(O)CH₃. When the term amido is used with the “substituted” modifier,it refers to groups, defined as —NHR, in which R is substituted acyl, asthat term is defined above. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The term “alkylideneamino” when used without the “substituted” modifierrefers to the group —N═R_(a), in which R_(a) is an alkylidene, as thatterm is defined above. The term “substituted alkylideneamino” refers tothe group N═R_(a), in which R_(a) is a substituted alkylidene, as thatterm is defined above.

The term “aralkylideneamino” when used without the “substituted”modifier refers to the group —N═R_(a), in which R_(a) is anaralkylidene, as that term is defined above. The term “substitutedaralkylideneamino” refers to the group N═R_(a), in which R_(a) is asubstituted aralkylidene, as that term is defined above.

The term “alkylaminodiyl” when used without the “substituted” modifierrefers to a non-aromatic divalent group, wherein the alkylaminodiylgroup is attached with two σ-bonds, with (a) two saturated carbon atomsas points of attachment, (b) one saturated carbon atom and one nitrogenatom as points of attachment, or (c) two nitrogen atoms as points ofattachment, further having a linear or branched, cyclo, cyclic oracyclic structure, no double or triple bonds in the group's backbone,further having no backbone atoms other than carbon or nitrogen andhaving at least one of each of these atoms in the group's backbone, andno side chains comprising groups other than hydrogen or alkyl. Thegroups, —NH—CH₂CH₂—, —CH₂—NH—CH₂CH₂—, —NH—CH₂CH₂—NH— and —NH—CH₂—NH— arenon-limiting examples of alkylaminodiyl groups. The term “substitutedalkylaminodiyl” refers to a divalent group, wherein the substitutedalkylaminodiyl group is attached with two σ-bonds, with (a) twosaturated carbon atoms as points of attachment, (b) one saturated carbonatom and one nitrogen atom as points of attachment, or (c) two nitrogenatoms as points of attachment, further having a linear or branched,cyclo, cyclic or acyclic structure, no carbon-carbon double or triplebonds in the group's backbone, and at least one atom independentlyselected from the group consisting of O, F, Cl, Br, I, Si, P, and S, orhaving additional nitrogen atom beyond those in the group's backbone.The following groups are non-limiting example of a substitutedalkylaminodiyl groups: —NH—CH₂C(OH)H—NH— and —NH—CH₂C(Cl)H—CH₂—.

The term “alkenylaminodiyl” when used without the “substituted” modifierrefers to a divalent group that is nonaromatic prior to attachment,wherein the alkenylaminodiyl group is attached with two σ-bonds, whichmay become aromatic upon attachment, with (a) two carbon atoms as pointsof attachment, (b) one carbon atom and one nitrogen atom as points ofattachment, or (c) two nitrogen atoms as points of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon double bond or carbon-nitrogen double that isnon-aromatic at least prior to attachment, further having no backboneatoms other than carbon or nitrogen, and no side chains comprisinggroups other than hydrogen or alkyl. The groups, —NH—CH═CH—, —NH—CH═N—and —NH—CH═CH—NH— are non-limiting examples of alkenylaminodiyl groups.The term “substituted alkenylaminodiyl” refers to a divalent group thatis nonaromatic prior to attachment, wherein the substitutedalkenylaminodiyl group is attached with two σ-bonds, which may becomearomatic upon attachment, with (a) two carbon atoms as points ofattachment, (b) one carbon atom and one nitrogen atom as points ofattachment, or (c) two nitrogen atoms as points of attachment, furtherhaving a linear or branched, cyclo, cyclic or acyclic structure, atleast one carbon-carbon double bond or carbon nitrogen double bond thatis non-aromatic at least prior to attachment and at least one atomindependently selected from the group consisting of O, F, Cl, Br, I, Si,P, and S, or having additional nitrogen atoms beyond those in thegroup's backbone. The following groups are non-limiting example of asubstituted alkenylaminodiyl groups: —NH—CH═C(OH)—CH₂— and N═CHC(Cl)H—.

The term “alkenylaminooxydiyl” when used without the “substituted”modifier refers to a divalent group, wherein the alkenylaminooxydiylgroup is attached with two σ-bonds, which may become aromatic uponattachment, with two atoms selected from the group consisting of carbon,oxygen and nitrogen as points of attachment, further having a linear orbranched, cyclo, cyclic or acyclic structure, at least one carbon-carbondouble bond, carbon-nitrogen double, or nitrogen-nitrogen double bondthat is non-aromatic at least prior to attachment, further having nobackbone atoms other than carbon nitrogen or oxygen and having at leastone of each of these three atoms in the backbone, and no side chainscomprising groups other than hydrogen or alkyl. The group —O—CH═N—, is anon-limiting example of an alkenylaminooxydiyl group. The term“substituted alkenylaminooxydiyl” refers to a divalent group that isattached with two σ-bonds, which may become aromatic upon attachmentwith two atoms selected from the group consisting of carbon, oxygen andnitrogen as points of attachment, further having a linear or branched,cyclo, cyclic or acyclic structure, at least one carbon-carbon doublebond or carbon nitrogen double bond that is non-aromatic at least priorto attachment and at least one atom independently selected from thegroup consisting of F, Cl, Br, I, Si, P, and S, or having one or moreadditional nitrogen and/or oxygen atoms beyond those in the group'sbackbone. The following groups are non-limiting example of a substitutedalkenylaminooxydiyl groups: —NH—CH═C(OH)—O— and —N═CHC(Cl)H—O—.

The term “alkenylaminothiodiyl” when used without the “substituted”modifier refers to a divalent group that is nonaromatic prior toattachment, wherein the alkenylaminothiodiyl group is attached with twoσ-bonds, which may become aromatic upon attachment, with two atomsselected from the group consisting of carbon, nitrogen and sulfur aspoints of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, at least one carbon-carbon double bond,carbon-nitrogen double, or nitrogen-nitrogen double bond that isnon-aromatic at least prior to attachment, further having no backboneatoms other than carbon, nitrogen or sulfur and having at least one ofeach of these three atoms in the backbone, and no side chains comprisinggroups other than hydrogen or alkyl. The group —S—CH═N—, is anon-limiting example of an alkenylaminothiodiyl group. The term“substituted alkenylaminothiodiyl” refers to a divalent group that isattached with two σ-bonds, which may become aromatic upon attachmentwith two atoms selected from the group consisting of carbon, nitrogenand sulfur as points of attachment, further having a linear or branched,cyclo, cyclic or acyclic structure, at least one carbon-carbon doublebond or carbon nitrogen double bond that is non-aromatic at least priorto attachment and at least one atom independently selected from thegroup consisting of O, F, Cl, Br, I, Si, and P, or having one or moreadditional nitrogen and/or sulfur atoms beyond those in the group'sbackbone. The following groups are non-limiting example of a substitutedalkenylaminothiodiyl groups: —NH—CH═C(OH)—S— and —N═CHC(Cl)H—S—.

The term “alkylimino” when used without the “substituted” modifierrefers to the group ═NR, wherein the alkylimino group is attached withone σ-bond and one π-bond, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylimino groups include: ═NCH,═NCH₂CH₃ and ═N-cyclohexyl. The term “substituted alkylimino” refers tothe group ═NR, wherein the alkylimino group is attached with one σ-bondand one π-bond, in which R is a substituted alkyl, as that term isdefined above. For example, ═NCH₂CF₃ is a substituted alkylimino group.

Similarly, the terms “alkenylimino”, “alkynylimino”, “arylimino”,“aralkylimino”, “heteroarylimino”, “heteroaralkylimino” and “acylimino”,when used without the “substituted” modifier, refers to groups, definedas ═NR, wherein the alkylimino group is attached with one σ-bond and oneπ-bond, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylimino, alkynylimino, arylimino,aralkylimino and acylimino is modified by “substituted,” it refers tothe group ═NR, wherein the alkylimino group is attached with one σ-bondand one π-bond, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl, heteroaralkyl and acyl, respectively.

The term “fluoroalkyl” when used without the “substituted” modifierrefers to an alkyl, as that term is defined above, in which one or morefluorines have been substituted for hydrogens. The groups, —CH₂F, —CF₂H,—CF₃, and —CH₂CF₃ are non-limiting examples of fluoroalkyl groups. Theterm “substituted fluoroalkyl” refers to a non-aromatic monovalent groupwith a saturated carbon atom as the point of attachment, a linear orbranched, cyclo, cyclic or acyclic structure, at least one fluorineatom, no carbon-carbon double or triple bonds, and at least one atomindependently selected from the group consisting of N, O, Cl, Br, I, Si,P, and S. The following group is a non-limiting example of a substitutedfluoroalkyl: —CFHOH.

The term “alkylphosphate” when used without the “substituted” modifierrefers to the group —OP(O)(OH)(OR), in which R is an alkyl, as that termis defined above. Non-limiting examples of alkylphosphate groupsinclude: —OP(O)(OH)(OMe) and —OP(O)(OH)(OEt). The term “substitutedalkylphosphate” refers to the group —OP(O)(OH)(OR), in which R is asubstituted alkyl, as that term is defined above.

The term “dialkylphosphate” when used without the “substituted” modifierrefers to the group —OP(O)(OR)(OR′), in which R and R′ can be the sameor different alkyl groups, or R and R′ can be taken together torepresent an alkanediyl having two or more saturated carbon atoms, atleast two of which are attached via the oxygen atoms to the phosphorusatom. Non-limiting examples of dialkylphosphate groups include:—OP(O)(OMe)₂, —OP(O)(OEt)(OMe) and —OP(O)(OEt)₂. The term “substituteddialkylphosphate” refers to the group —OP(O)(OR)(OR′), in which R and R′can be the same or different substituted alkyl groups, one of R or R′ isan alkyl and the other is a substituted alkyl, or R and R′ can be takentogether to represent a substituted alkanediyl with two or moresaturated carbon atoms, at least two of which are attached via theoxygen atoms to the phosphorous.

The term “alkylthio” when used without the “substituted” modifier refersto the group —SR, in which R is an alkyl, as that term is defined above.Non-limiting examples of alkylthio groups include: —SCH₃, —SCH₂CH₃,—SCH₂CH₂CH₃, —SCH(CH₃)₂, —SCH(CH₂)₂, —S-cyclopentyl, and —S-cyclohexyl.The term “substituted alkylthio” refers to the group —SR, in which R isa substituted alkyl, as that term is defined above. For example,—SCH₂CF₃ is a substituted alkylthio group.

Similarly, the terms “alkenylthio”, “alkynylthio”, “arylthio”,“aralkylthio”, “heteroarylthio”, “heteroaralkylthio”, and “acylthio”,when used without the “substituted” modifier, refers to groups, definedas —SR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,heteroaralkyl and acyl, respectively, as those terms are defined above.When any of the terms alkenylthio, alkynylthio, arylthio, aralkylthio,heteroarylthio, heteroaralkylthio, and acylthio is modified by“substituted,” it refers to the group —SR, in which R is substitutedalkenyl, alkynyl, aryl, aralkyl, heteroaryl, heteroaralkyl and acyl,respectively.

The term “thioacyl” when used without the “substituted” modifier refersto a monovalent group with a carbon atom of a thiocarbonyl group as thepoint of attachment, further having a linear or branched, cyclo, cyclicor acyclic structure, further having no additional atoms that are notcarbon or hydrogen, beyond the sulfur atom of the carbonyl group. Thegroups, —CHS, —C(S)CH₃, —C(S)CH₂CH₃, —C(S)CH₂CH₂CH₃, —C(S)CH(CH₃)₂,—C(S)CH(CH₂)₂, —C(S)C₆H₅, —C(S)C₆H₄CH₃, —C(S)C₆H₄CH₂CH₃,—C(S)C₆H₃(CH₃)₂, and —C(S)CH₂C₆H₅, are non-limiting examples of thioacylgroups. The term “thioacyl” therefore encompasses, but is not limitedto, groups sometimes referred to as “alkyl thiocarbonyl” and “arylthiocarbonyl” groups. The term “substituted thioacyl” refers to aradical with a carbon atom as the point of attachment, the carbon atombeing part of a thiocarbonyl group, further having a linear or branched,cyclo, cyclic or acyclic structure, further having at least one atom, inaddition to the sulfur atom of the carbonyl group, independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.The groups, —C(S)CH₂CF₃, —C(S)O₂H, —C(S)OCH₃, —C(S)OCH₂CH₃,—C(S)OCH₂CH₂CH₃, —C(S)OC₆H₅, —C(S)OCH(CH₃)₂, —C(S)OCH(CH₂)₂, —C(S)NH₂,and —C(S)NHCH₃, are non-limiting examples of substituted thioacylgroups. The term “substituted thioacyl” encompasses, but is not limitedto, “heteroaryl thiocarbonyl” groups.

The term “alkylsulfonyl” when used without the “substituted” modifierrefers to the group —S(O)₂R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfonyl groups include:—S(O)₂CH₃, —S(O)₂CH₂CH₃, —S(O)₂CH₂CH₂CH₃, —S(O)₂CH(CH₃)₂,—S(O)₂CH(CH₂)₂, —S(O)₂-cyclopentyl, and —S(O)₂-cyclohexyl. The term“substituted alkylsulfonyl” refers to the group —S(O)₂R, in which R is asubstituted alkyl, as that term is defined above. For example,—S(O)₂CH₂CF₃ is a substituted alkylsulfonyl group.

Similarly, the terms “alkenylsulfonyl”, “alkynylsulfonyl”,“arylsulfonyl”, “aralkylsulfonyl”, “heteroarylsulfonyl”, and“heteroaralkylsulfonyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)₂R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfonyl,alkynylsulfonyl, arylsulfonyl, aralkylsulfonyl, heteroarylsulfonyl, andheteroaralkylsulfonyl is modified by “substituted,” it refers to thegroup —S(O)₂R, in which R is substituted alkenyl, alkynyl, aryl,aralkyl, heteroaryl and heteroaralkyl, respectively.

The term “alkylsulfinyl” when used without the “substituted” modifierrefers to the group —S(O)R, in which R is an alkyl, as that term isdefined above. Non-limiting examples of alkylsulfinyl groups include:—S(O)CH₃, —S(O)CH₂CH₃, —S(O)CH₂CH₂CH₃, —S(O)CH(CH₃)₂, —S(O)CH(CH₂)₂,—S(O)-cyclopentyl, and —S(O)-cyclohexyl. The term “substitutedalkylsulfinyl” refers to the group —S(O)R, in which R is a substitutedalkyl, as that term is defined above. For example, —S(O)CH₂CF₃ is asubstituted alkylsulfinyl group.

Similarly, the terms “alkenylsulfinyl”, “alkynylsulfinyl”,“arylsulfinyl”, “aralkylsulfinyl”, “heteroarylsulfinyl”, and“heteroaralkylsulfinyl” when used without the “substituted” modifier,refers to groups, defined as —S(O)R, in which R is alkenyl, alkynyl,aryl, aralkyl, heteroaryl, and heteroaralkyl, respectively, as thoseterms are defined above. When any of the terms alkenylsulfinyl,alkynylsulfinyl, arylsulfinyl, aralkylsulfinyl, heteroarylsulfinyl, andheteroaralkylsulfinyl is modified by “substituted,” it refers to thegroup —S(O)R, in which R is substituted alkenyl, alkynyl, aryl, aralkyl,heteroaryl and heteroaralkyl, respectively.

The term “alkylammonium” when used without the “substituted” modifierrefers to a group, defined as —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, in which R,R′ and R″ are the same or different alkyl groups, or any combination oftwo of R, R′ and R″ can be taken together to represent an alkanediyl.Non-limiting examples of alkylammonium cation groups include:—NH₂(CH₃)⁺, —NH₂(CH₂CH₃)⁺, —NH₂(CH₂CH₂CH₃)⁺, —NH(CH₃)₂ ⁺, —NH(CH₂CH₃)₂⁺, —NH(CH₂CH₂CH₃)₂ ⁺, —N(CH₃)₃ ⁺, —N(CH₃)(CH₂CH₃)₂ ⁺, —N(CH₃)₂(CH₂CH₃)⁺,—NH₂C(CH₃)₃ ⁺, —NH(cyclopentyl)₂ ⁺, and —NH₂(cyclohexyl)⁺. The term“substituted alkylammonium” refers —NH₂R⁺, —NHRR′⁺, or —NRR′R″⁺, inwhich at least one of R, R′ and R″ is a substituted alkyl or two of R,R′ and R″ can be taken together to represent a substituted alkanediyl.When more than one of R, R′ and R″ is a substituted alkyl, they can bethe same of different. Any of R, R′ and R″ that are not eithersubstituted alkyl or substituted alkanediyl, can be either alkyl, eitherthe same or different, or can be taken together to represent aalkanediyl with two or more carbon atoms, at least two of which areattached to the nitrogen atom shown in the formula.

The term “alkylsulfonium” when used without the “substituted” modifierrefers to the group —SRR′⁺, in which R and R′ can be the same ordifferent alkyl groups, or R and R′ can be taken together to representan alkanediyl. Non-limiting examples of alkylsulfonium groups include:—SH(CH₃)⁺, —SH(CH₂CH₃)⁺, —SH(CH₂CH₂CH₃)⁺, —S(CH₃)₂ ⁺, —S(CH₂CH₃)₂ ⁺,—S(CH₂CH₂CH₃)₂ ⁺, —SH(cyclopentyl)⁺, and —SH(cyclohexyl)⁺. The term“substituted alkylsulfonium” refers to the group —SRR′⁺, in which R andR′ can be the same or different substituted alkyl groups, one of R or R′is an alkyl and the other is a substituted alkyl, or R and R′ can betaken together to represent a substituted alkanediyl. For example,—SH(CH₂CF₃)⁺ is a substituted alkylsulfonium group.

The term “alkylsilyl” when used without the “substituted” modifierrefers to a monovalent group, defined as —SiH₂R, —SiHRR′, or —SiRR′R″,in which R, R′ and R″ can be the same or different alkyl groups, or anycombination of two of R, R′ and R″ can be taken together to represent analkanediyl. The groups, —SiH₂CH₃, —SiH(CH₃)₂, —Si(CH₃)₃ and—Si(CH₃)₂C(CH₃)₃, are non-limiting examples of unsubstituted alkylsilylgroups. The term “substituted alkylsilyl” refers to —SiH₂R, —SiHRR′, or—SiRR′R″, in which at least one of R, R′ and R″ is a substituted alkylor two of R, R′ and R″ can be taken together to represent a substitutedalkanediyl. When more than one of R, R′ and R″ is a substituted alkyl,they can be the same of different. Any of R, R′ and R″ that are noteither substituted alkyl or substituted alkanediyl, can be either alkyl,either the same or different, or can be taken together to represent aalkanediyl with two or more saturated carbon atoms, at least two ofwhich are attached to the silicon atom.

In addition, atoms making up the compounds of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present invention may be replaced bya silicon atom(s). Furthermore, it is contemplated that one or moreoxygen atom(s) of a compound of the present invention may be replaced bya sulfur or selenium atom(s).

A compound having a formula that is represented with a dashed bond isintended to include the formulae optionally having zero, one or moredouble bonds. Thus, for example, the structure

includes the structures

As will be understood by a person of skill in the art, no one such ringatom forms part of more than one double bond.

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

As used herein, a “chiral auxiliary” refers to a removable chiral groupthat is capable of influencing the stereoselectivity of a reaction.Persons of skill in the art are familiar with such compounds, and manyare commercially available.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical SaltsProperties, and Use (2002).

As used herein, “predominantly one enantiomer” means that a compoundcontains at least about 85% of one enantiomer, or more preferably atleast about 90% of one enantiomer, or even more preferably at leastabout 95% of one enantiomer, or most preferably at least about 99% ofone enantiomer. Similarly, the phrase “substantially free from otheroptical isomers” means that the composition contains at most about 15%of another enantiomer or diastereomer, more preferably at most about 10%of another enantiomer or diastereomer, even more preferably at mostabout 5% of another enantiomer or diastereomer, and most preferably atmost about 1% of another enantiomer or diastereomer.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an inhibitor according to the present invention. The prodrug itselfmay or may not also have activity with respect to a given targetprotein. For example, a compound comprising a hydroxy group may beadministered as an ester that is converted by hydrolysis in vivo to thehydroxy compound. Suitable esters that may be converted in vivo intohydroxy compounds include acetates, citrates, lactates, phosphates,tartrates, malonates, oxalates, salicylates, propionates, succinates,fumarates, maleates, methylene-bis-β-hydroxynaphthoate, gentisates,isethionates, di-p-toluoyltartrates, methanesulfonates,ethanesulfonates, benzenesulfonates, p-toluenesulfonates,cyclohexylsulfamates, quinates, esters of amino acids, and the like.Similarly, a compound comprising an amine group may be administered asan amide that is converted by hydrolysis in vivo to the amine compound.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

The invention contemplates that for any stereocenter or axis ofchirality for which stereochemistry has not been defined, thatstereocenter or axis of chirality can be present in its R form, S form,or as a mixture of the R and S forms, including racemic and non-racemicmixtures.

“Substituent convertible to hydrogen in vivo” means any group that isconvertible to a hydrogen atom by enzymological or chemical meansincluding, but not limited to, hydrolysis and hydrogenolysis. Examplesinclude hydrolyzable groups, such as acyl groups, groups having anoxycarbonyl group, amino acid residues, peptide residues,o-nitrophenylsulfenyl, trimethylsilyl, tetrahydro-pyranyl,diphenylphosphinyl, and the like. Examples of acyl groups includeformyl, acetyl, trifluoroacetyl, and the like. Examples of groups havingan oxycarbonyl group include ethoxycarbonyl, tert-butoxycarbonyl(—C(O)OC(CH₃)₃), benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,vinyloxycarbonyl, β-(p-toluenesulfonyl)ethoxycarbonyl, and the like.Suitable amino acid residues include, but are not limited to, residuesof Gly (glycine), Ala (alanine), Arg (arginine), Asn (asparagine), Asp(aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine),Ile (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe(phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp(tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse(homoserine), 4-Hyp (4-hydroxyproline), 5-Hyl (5-hydroxylysine), Orn(ornithine) and β-Ala. Examples of suitable amino acid residues alsoinclude amino acid residues that are protected with a protecting group.Examples of suitable protecting groups include those typically employedin peptide synthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Suitable peptide residues include peptide residuescomprising two to five, and optionally amino acid residues. The residuesof these amino acids or peptides can be present in stereochemicalconfigurations of the D-form, the L-form or mixtures thereof. Inaddition, the amino acid or peptide residue may have an asymmetriccarbon atom. Examples of suitable amino acid residues having anasymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val,Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbonatom include peptide residues having one or more constituent amino acidresidues having an asymmetric carbon atom. Examples of suitable aminoacid protecting groups include those typically employed in peptidesynthesis, including acyl groups (such as formyl and acetyl),arylmethyloxycarbonyl groups (such as benzyloxycarbonyl andp-nitrobenzyloxycarbonyl), tert-butoxycarbonyl groups (—C(O)OC(CH₃)₃),and the like. Other examples of substituents “convertible to hydrogen invivo” include reductively eliminable hydrogenolyzable groups. Examplesof suitable reductively eliminable hydrogenolyzable groups include, butare not limited to, arylsulfonyl groups (such as o-toluenesulfonyl);methyl groups substituted with phenyl or benzyloxy (such as benzyl,trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such asbenzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); andhaloethoxycarbonyl groups (such as β,β,β-trichloroethoxycarbonyl andβ-iodoethoxycarbonyl).

“Therapeutically effective amount” or “pharmaceutically effectiveamount” means that amount which, when administered to a subject orpatient for treating a disease, is sufficient to effect such treatmentfor the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

Other abbreviations used herein are as follows: DMSO, dimethylsulfoxide; NO, nitric oxide; iNOS, inducible nitric oxide synthase;COX-2, cyclooxygenase-2; NGF, nerve growth factor; IBMX,isobutylmethylxanthine; FBS, fetal bovine serum; GPDH, glycerol3-phosphate dehydrogenase; RXR, retinoid X receptor; TGF-β, transforminggrowth factor-β; IFNγ or IFN-γ, interferon-γ; LPS, bacterial endotoxiclipopolysaccharide; TNFα or TNF-α, tumor necrosis factor-α; IL-1β,interleukin-1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TCA,trichloroacetic acid; HO-1, inducible heme oxygenase.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

II. Monocyclic Cyano Enones

Novel monocyclic cyano enones (MCEs) were synthesized and their Michaelreactivity and biological activity were tested in order to identifyuseful fragments for drug discovery. As part of these investigations,MCE-1 was found to be a highly reactive Michael acceptor. MCE-1 givesreversible and selective Michael adducts with thiol nucleophiles. MCE-1is also highly potent for inhibition of NO production in RAW cellsstimulated by interferon-γ (iNOS assay) and induction of the phase 2response, specifically, on elevation of NAD(P)H-quinone oxidoreductasein Hepa1c1c7 cells (NQO1 assay).

The potency of MCE-1 was found to be higher than CDDO in the iNOS assay.A homology model of the IKKβ based on the X-ray structure of thecheckpoint-2 kinase has been generated and employing it binding modelsof MCE-1 with Cys¹⁷⁹ of IKKβ examined. Without being bound by theory ormechanism, these studies are consistent with our belief that ligandscomprising the MCE-1 pharmacore, the A-ring of CDDO and TBE-31, willreversibly and selectively act on Cys¹⁷⁹ in the kinase domain on IKKβ.This series of new ligands provide unique IKKβ inhibitors which areATP-non-competitive. The compounds disclosed herein may also be used tointeract with a number of different protein targets including JAK1 andKeap1.

A. Synthesis

MCE-1, 3, and 4 were synthesized by the sequence shown in Scheme 2(Example 2). Compound 2 was prepared in 99% yield by ketalization ofcommercially available 4-oxocyclohexanonecarboxylic acid ethyl ester (1)(Phansavath et al., 1998). Nucleophilic addition of the enolate of 2 toiodomethane gave 3 in 85% yield. Reduction of 3 with LiAlH₄, followed byoxidation with CrO₃ afforded 5 (94% yield). Wittig reaction of 5 with(chloro-methyl)triphenylphosphonium chloride (Mella et al., 1988),followed by dehydrochlorination with MeLi and subsequent treatment withchlorotrimethylsilane (TMSC1) (Corey et al., 1973) produced 6 in 71%yield. The ketal of 6 was removed under acidic conditions to yield 7 in98% yield. Enone 8 was prepared by addition of phenylselenyl group tolithium enolate of 7 and subsequent oxidation/elimination with 30%aqueous hydrogen peroxide (36% yield). Formylation of 8 with ethylformate, followed by the condensation with hydroxylamine, gave isoxazole9 in 94% yield. The cleavage of the isoxiazole ring of 9 under basicconditions produced MCE-4 (5,6-dihydro-MCE-1, dhMCE-1) in quantitativeyield. MCE-1 was obtained from MCE-4 (dhMCE-1) by DDQ oxidation (47%yield). Compound 10 was prepared in 78% yield from 7 by cyanation withp-toluenesulfonyl cyanide (p-TsCN, Kahne et al., 1981), followed by DDQoxidation. Removal of TMS group from 10 afforded MCE-3 in 50% yield.MCE-1 itself would be synthesized in four steps from a known compound,4-ethynyl-4-methylcyclohex-2-en-1-one (Semmelhack et al., 1993) by thesame sequence as for MCE-1 from 8.

Known compound MCE-2 was prepared from 4,4-dimethylcyclohex-2-enoneaccording to the methods in the literature (Liu et al., 2000, which isincorporated herein by reference), as summarized here:

(a) HCO₂Et, NaOMe, PhH; (b) NH₂OH.HCl, aqueous EtOH; (c) NaOMe, MeOH,Et₂O; (d) DDQ, PhH.

New compound, MCE-5 was synthesized in 5 steps from 14 (Scheme 3,Example 3), which was prepared by Robinson annulation withisobutyraldehyde and ethyl vinyl ketone (Paquette et al. 1989, which isincorporated herein by reference). Known compound 15 was prepared in 50%yield by reductive methylation of 14 (Smith et al., 1967, which isincorporated herein by reference). Formylation of 15, followed by thetreatment with hydroxylamine, gave 17 in 77% yield. The cleavage of theisoxazole of 17 with sodium methoxide afforded dhMCE-5 in 90% yield.MCE-5 was obtained by DDQ oxidation of dhMCE-5 in 23% yield.

New compound, MCE-15 was synthesized from 5 by the sequence shown inScheme 4 (Example 4). Wittig reaction of 5 withmethyltriphenylphosphonium iodide, followed by deketalization, gave 19in 86% yield. Enone 20 was obtained as an inseparable mixture of 20 and19 (mole ratio 1.4:1) by addition of phenylselenyl group to lithiumenolate of 19 and subsequent oxidation/elimination with 30% aqueoushydrogen peroxide. Formylation of the mixture with ethyl formateafforded a mixture of 21 and 22. Compound 21 was isolated from themixture by flash chromatography. MCE-15 was prepared from 21 by the samesequence as for MCE-2 (see above).

MCE-1 analogue, MCE-13 having a benzyl group was synthesized by thesequence shown in Scheme 5 (Example 5). Nucleophilic addition of theenolate of 2 to benzyl bromide gave 25 in 81% yield. Reduction of 25,followed by oxidation produced 27 in 59% yield. The aldehyde 27 wasconverted to 28 using the Bestmann-Ohira reagent(dimethyl-1-diazo-2-oxopropylphosphonate) and potassium carbonate (85%yield, Müller, et al., 1996). The ethynyl group of 28 was protected byTMS group to give 29 in 89% yield. MCE-13 was obtained in 3.4% yieldfrom 29 by the same sequence as for MCE-1 from 6 (see Scheme 2 below).

MCE-7, tricycle containing MCE-1 as a fragment, was synthesized fromknown compound 33 (Honda et al., 2007) by the sequence shown in Scheme6. The ethynyl group of 33 was protected by TMS group to give 34 in 89%yield. A chromium-mediated allylic oxidation (Muzart, 1987) afforded 35in 47% yield. Compound 36 was synthesized in 57% yield by cyanation of35 with LDA and p-TsCN, followed by DDQ oxidation. Deketalization of 36under acidic conditions gave 37 in 86% yield. The TMS group was removedby tetra-(n-butyl) ammonium fluoride (TBAF) to give MCE-7 in 81% yield.

The compounds of the present disclosure were made using the methodsoutlined above and in the Examples section below. These methods can befurther modified and optimized using the principles and techniques oforganic chemistry as applied by a person skilled in the art. Suchprinciples and techniques are taught, for example, in March's AdvancedOrganic Chemistry: Reactions, Mechanisms, and Structure (2007), which isincorporated by reference herein.

B. UV Studies Using DTT and Keap1

MCE-1 gives the second UV absorption at a high wavelength (around 334nm) according to the addition of dithiothreitol (DTT) under the dilute(0.1 mM of MCE-1) and neutral aqueous conditions (pH 7.4 phosphatebuffered saline 1% ethanol), which are similar to the physiologicalconditions (see FIG. 1A). The absorption of MCE-1 at high wavelength,which increases according to the addition of DTT, is identical with theabsorption of MCE-4 (dhMCE-1) (see FIG. 1D) at high wavelength (around330 nm), which increases by the addition of NaOH (presumably due toenhanced H-bonding to the enol hydroxyl). This observation is consistentwith the second absorption of MCE-1 being derived from the Michaeladduct of DTT with MCE-1, because the chromophore of the Michael adductshould be the same as the enol form of MCE-4 (dhMCE-1), which shows theabsorption at higher wavelength than the keto form of MCE-4 (dhMCE-1).

Applicants note that the UV spectrum of MCE-1 (c=0.1 mM) in pH 7.4phosphate buffer solution shows two absorptions at 230 nm (log ε=3.96)and 322 nm (log ε=2.92). However, in ethanol and deionized water, thelong wavelength band is not observed (FIGS. 2 and 3). The phosphatebuffer (pH 7.4) includes 1 mM KH₂PO₄, 5.6 mM Na₂HPO₄, and 154 mM NaCl.Although the phosphate and chloride anions are much weaker nucleophilesthan the SH group, because the concentration of NaCl is 1540 times morethan the concentration of DTT (0.1 mM), these results suggest that thatMCE-1 reacts with chloride anion to give a Michael adduct.

Once DTT (0.1 mM) is added to the buffer solution of MCE-1, the adductwith chloride disappears (322 nm) and the adduct with DTT appears (333nm) (see FIG. 1A), thereby providing evidence that MCE-1 gives selectiveMichael adduction with thiol nucleophiles. Applicants note that thisresult is obtained despite that the concentration of DTT is 1,540 timeslower than that of NaCl.

UV spectrum of MCE-5 shows a weak absorption at 275 nm upon the additionof 1.0 mM DTT, while no apparent change occurs upon addition of 0.1 mMDTT (FIG. 4). Applicants note that this absorption is consistent withthe absorption of dhMCE-5 at 265 nm which increases by the addition ofNaOH (FIG. 5), suggesting that the weak absorption detected for MCE-5with 1.0 nM DTT is derived from the Michael adduct of DTT with MCE-5.

MCE-2 and MCE-15 do not give the second absorption by the addition ofDTT under the same conditions as for MCE-1. See FIGS. 1B and 10, whichprovide the results for MCE-2 and 15. MCE-3 (acetylenic enolizable cyanoenone) does not change when 0.1 mM of DTT is added while they show smallred shift when 1 mM of DTT is added (FIG. 1C).

MCE-7 and 13 like MCE-1 show second absorptions at around 330 nm, whichare derived from Michael adducts with DTT. The absorbance of MCE-7 (FIG.11) is similarly intense as the corresponding absorption of MCE-1. Theabsorbance of MCE-13 is a bit weaker than the corresponding absorptionof MCE-1 (FIG. 12)

Keap1 is an electrophile sensor in the cellular Keap1-Nrf2-antioxidantresponse elements (ARE) phase II response pathway that regulates thetranscription of genes for cytoprotective enzymes such as HO-1 and NQO1.Keap1 is a thiol-rich protein possessing 25 cysteine residues, some ofwhich are highly reactive (Dinkova-Kostova et al., 2002, which isincorporated herein by reference). UV studies on MCEs with Keap1 areconsistent with those with DTT, with MCE-1 showing the highestreactivity of the group. That is MCE-1 showed the highest reactivityboth on a thiol-rich protein as well as on a small thiol nucleophiles,further confirming the hypothesis that MCE-1 selectively reacts withthiol nucleophiles.

C. ¹H-NMR Studies

Also disclosed herein are ¹H-NMR results providing further evidence thatMCE-1 gives a reversible Michael adduct with DTT. In the ¹H NMR (500MHz, DMSO-d₆ at 21° C.) of MCE-1, the olefinic protons H_(A), H_(B), andH_(C) are observed at δ 8.17 ppm (d, J=3 Hz), 7.21 ppm (dd, J=3 and 10Hz), and 6.35 ppm (d, J=10 Hz), respectively, while olefinic protonsH_(A), H_(B), and H_(c) of MCE-1 decrease, an enol proton [at δ 11.12ppm (brs)] and new olefinic protons H_(D) and H_(E) appear and increaseaccording to increasing amounts of DTT. See FIG. 13. These new protonsare consistent with adduct formation via Michael addition of DTT toMCE-1. The development of additional small signals suggests that asecond stereoisomeric adduct forms as well.

In further experiments, at elevated temperatures, although the enolproton and olefinic protons H_(D) and H_(E) of Michael adduct decrease,the olefinic protons H_(A), H_(B), and H_(C) of MCE-1 increase. See FIG.14. These observations are consistent with reversible Michael addition.Although Michael adducts of CDDO and MCE-1 with DTT are observed by NMRand UV, isolation has not been possible and may not be possible. Theseresults suggest that the conversion of the Michael adducts to CDDO andMCE-1 is fast (Couch et al., 2005, which is incorporated herein byreference).

D. Biological Activity

Compounds of the present disclosure have been tested for inhibition ofnitric oxide (NO) production in RAW cells stimulated by interferon-γ(iNOS assay) and induction of NAD(P)H-quinone oxidoreductase inHepa1c1c7 cells (NQO1 assay), both assay systems we have previouslyemployed. At the same time, CDDO and TBE-31 were tested as a positivecontrols in the same assays. The results of the iNOS assay are shown inTable 1. Notably, MCE-1 shows the highest potency amongst MCEs in bothassays. The potency in the iNOS assay is more than CDDO (pentacycle),whose methyl ester is currently evaluated in Phase II clinical trials asan anti-inflammatory drug. Also the potency correlates to theirreactivity as Michael acceptors based on the above-summarized UVstudies. However, although the reactivity of MCE-7 is similar to MCE-1(compare FIG. 1A with FIG. 11), it is inactive at a concentration of 300nM in the iNOS assay and less potent than MCE-1 in the NQO1 assay.

TABLE 1 Suppression of IFNγ-induced NO production. RAW (10 ng/ml IFNγ)Compound Structure IC₅₀ MCE-1

8 nM MCE-2

380 nM MCE-7

>300 nM MCE-15

300 nM CDDO

20 nM TBE-31

1 nM

III. Homology Model of IKKβ and Identification of Further MCE Candidates

A homology model of the IKKβ based on the X-ray structure of thecheckpoint-2 kinase (2CN5.ent) was generated, and the two pockets, onehydrophobic and one hydrophilic, surrounding Cys¹⁷⁹ were targeted toidentify further lead compounds. The proposed syntheses of thesecompounds (as well as others) are provided in the examples sectionbelow.

The homology model of IKKβ was built using SWISSMODEL and the X-raystructures of other related kinases (checkpoint-2 kinase, 2cn5.pdb)within the Protein Data Bank. The sequence comparison of the twoproteins has a sufficient sequence similarity (36% sequence identity) toprovide a good working model. The homology-based IKKβ dimer wassubjected to several cycles of energy minimization to remove initialstrain, using the AMBER force field within the InsightII (MolecularSimulations, Inc.) program. The complex was then placed centrally in acube of 12 nm and soaked with water (37,700 water molecules) and thenenergy minimized using a steepest descent algorithm. Extensive moleculardynamics (MD) simulations (at 300 K, with an integration time step of 2fs, and constant pressure of 1 bar) were carried out with the complexusing the GROMACS program (Berendsen et al., 1995, which is incorporatedherein by reference) Pentium III processors running Linux. Starting withthis initial homology model, an initial set of ligands for whichbiological activity (MCE-1, CDDO, TBE-31) was examined (see Table 1).

For example, the binding of MCE-1 to Cys¹⁷⁹ based on molecular dynamicssimulations and energy minimization suggests that two Gln (Q176/Q197)make up the binding pocket to the cyano/hydroxyl function, while L178,F182, and L194 make up the hydrophobic pocket.

Based on this approach, additional ligands may be rationally designed tofurther enhance binding, targeting the two pockets proximal to theCys¹⁷⁹. Examples of such ligands and their synthesis and/or proposedsynthesis is provided in the Examples section below. Additionally, two“non-rational” approaches can be utilized to further expand the pool ofpossible ligands (autodock, fragment tethering). All three of thesemethods can be used in an iterative procedure, along with the resultsfrom the biological assays, in order to optimize binding affinity.

Rational Structure Based Screening—Ligand designs and/or modificationscan be examined by molecular modeling and computer simulations. whichallows refinement and optimization of the physicochemical propertiesbefore undertaking the synthesis. INSIGHT II and Chimera (UCSF) areutilized for the viewing and interactive manipulation and building ofthe protein structures. For extensive homology modeling one can utilizeWHATIF (Vriend, 1990, which is incorporated herein by reference) orModeler. Procheck (Laskowski et al., 1996), which is incorporated hereinby reference will be used to examine the “health” of all of the proteinstructures. The protein structures are then refined using extensivemolecular dynamics simulations using the GROMACs or NAMD simulationprograms, within a fully solvated simulation cell.

Virtual screening through a fragment tethering strategy—Complementary tothe rational design “educated” screening approach, a de novo design ofanalogs via a fragment tethering strategy can also be undertaken. Thismethod involves growing substituents onto a pharmacophore core of thelead compound to generate a compound library for virtual screening,analyzing the binding pocket, and determining where a specificfunctional group might bind tightly to the useful sites in the pocket.

Subsequently, these chemical groups are virtually tethered to produce amolecular skeleton which is converted into a plausible bioorganicmolecule to be synthesized. The computer program, Discovery Studio,allows such a “fragment-tethering” strategy to grow substituents onto apharmacophore core of the lead compound to generate a compound libraryfor virtual screening. It offers the possibility of discovering novelinhibitory molecules without a potentially biased effort toward aspecific class of compounds in the existing libraries/databases.

For example, using the structure of MCE-1 docked to IKKβ as the corestructure, two sites of substitution can be examined with fragments ofdifferent bulkiness and stereoelectronic properties from an in-house“fragment library” selected for synthetic availability andpharmacological properties. The resulting “virtual” compounds can thenbe docked into the IKKβ pockets to prioritize the binding affinity.

AutoDock—Different ligand targets may also be identified by utilizingdocking to obtain starting structures/topological orientations that canthen be further refined with MD simulations. The docking may be carriedout with autodock4.0 (Scripps) using the homology model of the IKKβgenerated as described above. This rigid docking procedure will providemultiple starting structures for further refinement via MD simulations,which is important to account for conformational changes that may takeplace during binding, an aspect that is being increasingly appreciatedin computational drug design. In addition to the autodock, we haverecently begun using GOLD (version 2.0) and the associated libraries ofsmall molecules. The inventors will carry out GOLD docking to generatenovel molecular fragments that fit well into the binding pocketssurrounding Cys179, that can then be incorporated into the syntheticefforts detailed in Aim #2. The complexes obtained from both of thedocking procedures will be soaked with TIP4 water molecules and thenundergo extensive MD simulations using the GROMACS program. During theMD simulations both the protein and ligand will be allowed to undergoconformational changes, allowing for the generation of optimal fit. Wehave developed shell scripts to automatic conversion of the output filesfrom the docking procedures to these MD simulations.

A homology model of the IKKβ based on the X-ray structure of thecheckpoint-2 kinase (2CN5.ent) was generated, and the two pockets, onehydrophobic and one hydrophilic, surrounding Cys¹⁷⁹ were targeted togenerate identify further lead compounds. Based on the identifiedhydrophobic and hydrophilic pockets surrounding Cys179 of IKKβ and thatthe distance between Cys¹⁷⁹ and either the hydrophobic or hydrophilicpocket is both 7-8 Å, a preliminary set of compounds has been designed.The proposed synthesis of some of the compounds is provided in theExamples section below.

In certain embodiments, there is provided a hydrophobic series, forexample, with structures as defined by formulas I and II, with compounds38 and 39 as respective examples of each.

Without being bound by theory or mechanism, based on the informationgleaned from the IKKβ homology model, n=1-3 and m=1-2 would give areasonable length in some embodiments because the distance between thereactive site of the Michael acceptor and the phenyl group of thesemolecules is similar to the distance between Cys¹⁷⁹ and hydrophobicpocket (about 7-8 Å).

In certain embodiments, there is provided a hydrophilic series, forexample, with structures as defined by formulas III, with compounds 48as an example thereof

Without being bound by theory or mechanism, based on the informationgleaned from the IKKβ homology model, n=1-2 would provide a reasonablelength in some embodiments. A proposed synthesis for 48 and othercompounds of the hydrophilic series is provided in the Examples sectionbelow. Other examples hydrophilic groups includes compounds havinghydroxyl or carboxyl groups. See, e.g., Example 13.

Various compounds appropriately sized for either hydrophilic orhydrophobic pockets may be synthesized using Sonogashira coupling(Sonogashira et al., 1975), for example, between known compound 65(Phansavath et al., 1998, which is incorporated herein by reference) andan aromatic halide.

If one inserts, for example, a phenyl, a thiophenyl, or a furyl groups,the resulting compound of generalized formula IV would have ahydrophobic moiety attached. If once were instead to insert pyridinyland imidazolyl groups, the resulting compound of formula IV would havehydrophilic moiety attached. As thiazolyl and oxazolyl groups areneutral, they are hydrophobic. However, quaternary salts of both groupsare hydrophilic. Therefore, compounds IV having a thiazolyl or oxazolylgroup might show either hydrophobic or hydrophilic properties and mayvary depending on the conditions, for example, the environment within inliving cells or proximate to a living cell. Scheme A provides examplesof moieties with these different properties.

In addition to monocyclic cyanoenones, the invention also providesbicyclic and tricyclic cyanoenones. For example:

In some embodiments, such compounds made have both hydrophilic andhydrophobic groups, for example, compounds according to formula VII:

The syntheses of certain bicyclic cyanoenones of formula VII areproposed in Example 17 below. Examples of tricyclic cyanoenones areprovided in Examples 6-8, below.

Once synthesized these compounds can be further evaluated for thebiological activity, for example, through an in vitro IKKβ kinase assayusing a commercially available HTScan® IKKβ Kinase Assay Kit. A suitableprotocol for such an assay is described in Example 1 below.

IV. Diseases Associated with Inflammation and/or Oxidative Stress

Inflammation is a biological process that provides resistance toinfectious or parasitic organisms and the repair of damaged tissue.Inflammation is commonly characterized by localized vasodilation,redness, swelling, and pain, the recruitment of leukocytes to the siteof infection or injury, production of inflammatory cytokines such asTNF-α and IL-1, and production of reactive oxygen or nitrogen speciessuch as hydrogen peroxide, superoxide and peroxynitrite. In later stagesof inflammation, tissue remodeling, angiogenesis, and scar formation(fibrosis) may occur as part of the wound healing process. Under normalcircumstances, the inflammatory response is regulated and temporary andis resolved in an orchestrated fashion once the infection or injury hasbeen dealt with adequately. However, acute inflammation can becomeexcessive and life-threatening if regulatory mechanisms fail.Alternatively, inflammation can become chronic and cause cumulativetissue damage or systemic complications.

Many serious and intractable human diseases involve dysregulation ofinflammatory processes, including diseases such as cancer,atherosclerosis, and diabetes, which were not traditionally viewed asinflammatory conditions. In the case of cancer, the inflammatoryprocesses are associated with tumor formation, progression, metastasis,and resistance to therapy. Atherosclerosis, long viewed as a disorder oflipid metabolism, is now understood to be primarily an inflammatorycondition, with activated macrophages playing an important role in theformation and eventual rupture of atherosclerotic plaques. Activation ofinflammatory signaling pathways has also been shown to play a role inthe development of insulin resistance, as well as in the peripheraltissue damage associated with diabetic hyperglycemia. Excessiveproduction of reactive oxygen species and reactive nitrogen species suchas superoxide, hydrogen peroxide, nitric oxide, and peroxynitrite is ahallmark of inflammatory conditions. Evidence of dysregulatedperoxynitrite production has been reported in a wide variety of diseases(Szabo et al., 2007; Schulz et al., 2008; Forstermann, 2006; Pall,2007).

Autoimmune diseases such as rheumatoid arthritis, lupus, psoriasis, andmultiple sclerosis involve inappropriate and chronic activation ofinflammatory processes in affected tissues, arising from dysfunction ofself vs. non-self recognition and response mechanisms in the immunesystem. In neurodegenerative diseases such as Alzheimer's andParkinson's diseases, neural damage is correlated with activation ofmicroglia and elevated levels of pro-inflammatory proteins such asinducible nitric oxide synthase (iNOS). Chronic organ failure such asrenal failure, heart failure, and chronic obstructive pulmonary diseaseis closely associated with the presence of chronic oxidative stress andinflammation, leading to the development of fibrosis and eventual lossof organ function.

Many other disorders involve oxidative stress and inflammation inaffected tissues, including inflammatory bowel disease; inflammatoryskin diseases; mucositis related to radiation therapy and chemotherapy;eye diseases such as uveitis, glaucoma, macular degeneration, andvarious forms of retinopathy; transplant failure and rejection;ischemia-reperfusion injury; chronic pain; degenerative conditions ofthe bones and joints including osteoarthritis and osteoporosis; asthmaand cystic fibrosis; seizure disorders; and neuropsychiatric conditionsincluding schizophrenia, depression, bipolar disorder, post-traumaticstress disorder, attention deficit disorders, autism-spectrum disorders,and eating disorders such as anorexia nervosa. Dysregulation ofinflammatory signaling pathways is believed to be a major factor in thepathology of muscle wasting diseases including muscular dystrophy andvarious forms of cachexia.

A variety of life-threatening acute disorders also involve dysregulatedinflammatory signaling, including acute organ failure involving thepancreas, kidneys, liver, or lungs, myocardial infarction or acutecoronary syndrome, stroke, septic shock, trauma, severe burns, andanaphylaxis.

Many complications of infectious diseases also involve dysregulation ofinflammatory responses. Although an inflammatory response can killinvading pathogens, an excessive inflammatory response can also be quitedestructive and in some cases can be a primary source of damage ininfected tissues. Furthermore, an excessive inflammatory response canalso lead to systemic complications due to overproduction ofinflammatory cytokines such as TNF-α and IL-1. This is believed to be afactor in mortality arising from severe influenza, severe acuterespiratory syndrome, and sepsis.

The aberrant or excessive expression of either iNOS or cyclooxygenase-2(COX-2) has been implicated in the pathogenesis of many diseaseprocesses. For example, it is clear that NO is a potent mutagen (Tamirand Tannebaum, 1996), and that nitric oxide can also activate COX-2(Salvemini et al., 1994). Furthermore, there is a marked increase iniNOS in rat colon tumors induced by the carcinogen, azoxymethane(Takahashi et al., 1997). A series of synthetic triterpenoid analogs ofoleanolic acid have been shown to be powerful inhibitors of cellularinflammatory processes, such as the induction by IFN-γ of induciblenitric oxide synthase (iNOS) and of COX-2 in mouse macrophages. SeeHonda et al. (2000a); Honda et al. (2000b), and Honda et al. (2002),which are all incorporated herein by reference.

In one aspect, compounds disclosed herein are characterized by theirability to inhibit the production of nitric oxide in macrophage-derivedRAW 264.7 cells induced by exposure to γ-interferon. They are furthercharacterized by their ability to induce the expression of antioxidantproteins such as NQO1 and reduce the expression of pro-inflammatoryproteins such as COX-2 and inducible nitric oxide synthase (iNOS). Theseproperties are relevant to the treatment of a wide array of diseasesinvolving oxidative stress and dysregulation of inflammatory processesincluding cancer, mucositis resulting from radiation therapy orchemotherapy, autoimmune diseases, cardiovascular diseases includingatherosclerosis, ischemia-reperfusion injury, acute and chronic organfailure including renal failure and heart failure, respiratory diseases,diabetes and complications of diabetes, severe allergies, transplantrejection, graft-versus-host disease, neurodegenerative diseases,diseases of the eye and retina, acute and chronic pain, degenerativebone diseases including osteoarthritis and osteoporosis, inflammatorybowel diseases, dermatitis and other skin diseases, sepsis, burns,seizure disorders, and neuropsychiatric disorders.

Without being bound by theory, the activation of theantioxidant/anti-inflammatory Keap1/Nrf2/ARE pathway is believed to beimplicated in both the anti-inflammatory and anti-carcinogenicproperties of the compounds disclosed herein.

In another aspect, compounds disclosed herein may be used for treating asubject having a condition caused by elevated levels of oxidative stressin one or more tissues. Oxidative stress results from abnormally high orprolonged levels of reactive oxygen species such as superoxide, hydrogenperoxide, nitric oxide, and peroxynitrite (formed by the reaction ofnitric oxide and superoxide). The oxidative stress may be accompanied byeither acute or chronic inflammation. The oxidative stress may be causedby mitochondrial dysfunction, by activation of immune cells such asmacrophages and neutrophils, by acute exposure to an external agent suchas ionizing radiation or a cytotoxic chemotherapy agent (e.g.,doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion, by poor circulation or anemia, by localized orsystemic hypoxia or hyperoxia, by elevated levels of inflammatorycytokines and other inflammation-related proteins, and/or by otherabnormal physiological states such as hyperglycemia or hypoglycemia.

In animal models of many such conditions, stimulating expression ofinducible heme oxygenase (HO-1), a target gene of the Nrf2 pathway, hasbeen shown to have a significant therapeutic effect including models ofmyocardial infarction, renal failure, transplant failure and rejection,stroke, cardiovascular disease, and autoimmune disease (e.g., Sacerdotiet al., 2005; Abraham & Kappas, 2005; Bach, 2006; Araujo et al., 2003;Liu et al., 2006; Ishikawa et al., 2001; Kruger et al., 2006; Satoh etal., 2006; Zhou et al., 2005; Morse and Choi, 2005; Morse and Choi,2002). This enzyme breaks free heme down into iron, carbon monoxide(CO), and biliverdin (which is subsequently converted to the potentantioxidant molecule, bilirubin).

In another aspect, compounds of this invention may be used in preventingor treating tissue damage or organ failure, acute and chronic, resultingfrom oxidative stress exacerbated by inflammation. Examples of diseasesthat fall in this category include: heart failure, liver failure,transplant failure and rejection, renal failure, pancreatitis, fibroticlung diseases (cystic fibrosis and COPD, among others), diabetes(including complications), atherosclerosis, ischemia-reperfusion injury,glaucoma, stroke, autoimmune disease, autism, macular degeneration, andmuscular dystrophy. For example, in the case of autism, studies suggestthat increased oxidative stress in the central nervous system maycontribute to the development of the disease (Chauhan and Chauhan,2006).

Evidence also links oxidative stress and inflammation to the developmentand pathology of many other disorders of the central nervous system,including psychiatric disorders such as psychosis, major depression, andbipolar disorder; seizure disorders such as epilepsy; pain and sensorysyndromes such as migraine, neuropathic pain or tinnitus; and behavioralsyndromes such as the attention deficit disorders. See, e.g., Dickersonet al., 2007; Hanson et al., 2005; Kendall-Tackett, 2007; Lencz et al.,2007; Dudhgaonkar et al., 2006; Lee et al., 2007; Morris et al., 2002;Ruster et al., 2005; McIver et al., 2005; Sarchielli et al., 2006;Kawakami et al., 2006; Ross et al., 2003, which are all incorporated byreference herein. For example, elevated levels of inflammatorycytokines, including TNF, interferon-γ, and IL-6, are associated withmajor mental illness (Dickerson et al., 2007). Microglial activation hasalso been linked to major mental illness. Therefore, down-regulatinginflammatory cytokines and inhibiting excessive activation of microgliacould be beneficial in patients with schizophrenia, major depression,bipolar disorder, autism-spectrum disorders, and other neuropsychiatricdisorders.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively, inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of radiation therapy to a cancerpatient), or it may be administered therapeutically in settingsinvolving established oxidative stress and inflammation.

The compounds disclosed herein may be generally applied to the treatmentof inflammatory conditions, such as sepsis, dermatitis, autoimmunedisease and osteoarthritis. In one aspect, the compounds of thisinvention may be used to treat inflammatory pain and/or neuropathicpain, for example, by inducing Nrf2 and/or inhibiting NF-κB.

In one aspect, the compounds disclosed herein may be used to function asantioxidant inflammation modulators (AIMs) having potentanti-inflammatory properties that mimic the biological activity ofcyclopentenone prostaglandins (cyPGs). In one embodiment, the compoundsdisclosed herein may be used to control the production ofpro-inflammatory cytokines by selectively targeting regulatory cysteineresidues (RCRs) on proteins that regulate the transcriptional activityof redox-sensitive transcription factors. Activation of RCRs by cyPGs orAIMs has been shown to initiate a pro-resolution program in which theactivity of the antioxidant and cytoprotective transcription factor Nrf2is potently induced, and the activities of the pro-oxidant andpro-inflammatory transcription factors NF-κB and the STATs aresuppressed. This increases the production of antioxidant and reductivemolecules (e.g., NQO1, HO-1, SOD1, and/or γ-GCS) and/or decreasesoxidative stress and the production of pro-oxidant and pro-inflammatorymolecules (e.g., iNOS, COX-2, and/or TNF-α).

In some embodiments, the compounds disclosed herein may be used in thetreatment and prevention of diseases such as cancer, inflammation,Alzheimer's disease, Parkinson's disease, multiple sclerosis, autism,amyotrophic lateral sclerosis, autoimmune diseases such as rheumatoidarthritis, lupus, and MS, inflammatory bowel disease, all other diseaseswhose pathogenesis is believed to involve excessive production of eithernitric oxide or prostaglandins, and pathologies involving oxidativestress alone or oxidative stress exacerbated by inflammation.

Another aspect of inflammation is the production of inflammatoryprostaglandins such as prostaglandin E. These molecules promotevasodilation, plasma extravasation, localized pain, elevatedtemperature, and other symptoms of inflammation. The inducible form ofthe enzyme COX-2 is associated with their production, and high levels ofCOX-2 are found in inflamed tissues. Consequently, inhibition of COX-2may relieve many symptoms of inflammation and a number of importantanti-inflammatory drugs (e.g., ibuprofen and celecoxib) act byinhibiting COX-2 activity. Recent research, however, has demonstratedthat a class of cyclopentenone prostaglandins (cyPGs) (e.g., 15-deoxyprostaglandin J2, a.k.a. PGJ2) plays a role in stimulating theorchestrated resolution of inflammation (e.g., Rajakariar et al., 2007).COX-2 is also associated with the production of cyclopentenoneprostaglandins. Consequently, inhibition of COX-2 may interfere with thefull resolution of inflammation, potentially promoting the persistenceof activated immune cells in tissues and leading to chronic,“smoldering” inflammation. This effect may be responsible for theincreased incidence of cardiovascular disease in patients usingselective COX-2 inhibitors for long periods of time.

In one aspect, the compounds disclosed herein may be used to control theproduction of pro-inflammatory cytokines within the cell by selectivelyactivating regulatory cysteine residues (RCRs) on proteins that regulatethe activity of redox-sensitive transcription factors. Activation ofRCRs by cyPGs has been shown to initiate a pro-resolution program inwhich the activity of the antioxidant and cytoprotective transcriptionfactor Nrf2 is potently induced and the activities of the pro-oxidantand pro-inflammatory transcription factors NF-κB and the STATs aresuppressed. In some embodiments, this increases the production ofantioxidant and reductive molecules (NQO1, HO-1, SOD1, γ-GCS) anddecreases oxidative stress and the production of pro-oxidant andpro-inflammatory molecules (iNOS, COX-2, TNF-α). In some embodiments,the compounds of this invention may cause the cells that host theinflammatory event to revert to a non-inflammatory state by promotingthe resolution of inflammation and limiting excessive tissue damage tothe host.

E. Cancer

Further, the compounds of the present disclosure may be used to induceapoptosis in tumor cells, to induce cell differentiation, to inhibitcancer cell proliferation, to inhibit an inflammatory response, and/orto function in a chemopreventative capacity. For example, the inventionprovides new compounds that have one or more of the followingproperties: (1) an ability to induce apoptosis and differentiate bothmalignant and non-malignant cells, (2) an activity at sub-micromolar ornanomolar levels as an inhibitor of proliferation of many malignant orpremalignant cells, (3) an ability to suppress the de novo synthesis ofthe inflammatory enzyme inducible nitric oxide synthase (iNOS), (4) anability to inhibit NF-κB activation, and (5) an ability to induce theexpression of heme oxygenase-1 (HO-1).

The levels of iNOS and COX-2 are elevated in certain cancers and havebeen implicated in carcinogenesis and COX-2 inhibitors have been shownto reduce the incidence of primary colonic adenomas in humans (Rostom etal., 2007; Brown and DuBois, 2005; Crowel et al., 2003). iNOS isexpressed in myeloid-derived suppressor cells (MDSCs) (Angulo et al.,2000) and COX-2 activity in cancer cells has been shown to result in theproduction of prostaglandin E₂ (PGE₂), which has been shown to inducethe expression of arginase in MDSCs (Sinha et al., 2007). Arginase andiNOS are enzymes that utilize L-arginine as a substrate and produceL-ornithine and urea, and L-citrulline and NO, respectively. Thedepletion of arginine from the tumor microenvironment by MDSCs, combinedwith the production of NO and peroxynitrite has been shown to inhibitproliferation and induce apoptosis of T cells (Bronte et al., 2003).Inhibition of COX-2 and iNOS has been shown to reduce the accumulationof MDSCs, restore cytotoxic activity of tumor-associated T cells, anddelay tumor growth (Sinha et al., 2007; Mazzoni et al., 2002; Zhou etal., 2007).

Inhibition of the NF-κB and JAK/STAT signaling pathways has beenimplicated as a strategy to inhibit proliferation of cancer epithelialcells and induce their apoptosis. Activation of STAT3 and NF-κB has beenshown to result in suppression of apoptosis in cancer cells, andpromotion of proliferation, invasion, and metastasis. Many of the targetgenes involved in these processes have been shown to betranscriptionally regulated by both NF-κB and STAT3 (Yu et al., 2007).

In addition to their direct roles in cancer epithelial cells, NF-κB andSTAT3 also have important roles in other cells found within the tumormicroenvironment. Experiments in animal models have demonstrated thatNF-κB is required in both cancer cells and hematopoeitic cells topropagate the effects of inflammation on cancer initiation andprogression (Greten et al., 2004). NF-κB inhibition in cancer andmyeloid cells reduces the number and size, respectively, of theresultant tumors. Activation of STAT3 in cancer cells results in theproduction of several cytokines (IL-6, IL-10) which suppress thematuration of tumor-associated dendritic cells (DC). Furthermore, STAT3is activated by these cytokines in the dendritic cells themselvesInhibition of STAT3 in mouse models of cancer restores DC maturation,promotes antitumor immunity, and inhibits tumor growth (Kortylewski etal., 2005).

F. Treatment of Multiple Sclerosis

The compounds and methods of this invention may be used for treatingpatients for multiple sclerosis (MS). MS is known to be an inflammatorycondition of the central nervous system (Williams et al., 1994; Merrilland Benvenist, 1996; Genain and Nauser, 1997). Based on severalinvestigations, there is evidence suggesting that inflammatory,oxidative, and/or immune mechanisms are involved in the pathogenesis ofAlzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateralsclerosis (ALS), and MS (Bagasra et al., 1995; McGeer and McGeer, 1995;Simonian and Coyle, 1996; Kaltschmidt et al., 1997). Both reactiveastrocytes and activated microglia have been implicated in causation ofneurodegenerative disease (NDD) and neuroinflammatory disease (NID);there has been a particular emphasis on microglia as cells thatsynthesize both NO and prostaglandins as products of the respectiveenzymes, iNOS and COX-2. De novo formation of these enzymes may bedriven by inflammatory cytokines such as interferon-γ or interleukin-1.In turn, excessive production of NO may lead to inflammatory cascadesand/or oxidative damage in cells and tissues of many organs, includingneurons and oligodendrocytes of the nervous system, with consequentmanifestations in AD and MS, and possible PD and ALS (Coyle andPuttfarcken, 1993; Beal, 1996; Merrill and Benvenist, 1996; Simonian andCoyle, 1996; Vodovotz et al., 1996). Epidemiologic data indicate thatchronic use of NSAID's which block synthesis of prostaglandins fromarachidonate, markedly lower the risk for development of AD (McGeer etal., 1996; Stewart et al., 1997). Thus, agents that block formation ofNO and prostaglandins, may be used in approaches to prevention andtreatment of NDD. Successful therapeutic candidates for treating such adisease typically require an ability to penetrate the blood-brainbarrier. See, for example, U.S. Patent Publication 2009/0060873, whichis incorporated by reference herein in its entirety.

G. Neuroinflammation

Neuroinflammation encapsulates the idea that microglial and astrocyticresponses and actions in the central nervous system have a fundamentallyinflammation-like character, and that these responses are central to thepathogenesis and progression of a wide variety of neurologicaldisorders. This idea originated in the field of Alzheimer's disease(Griffin et al., 1989; Rogers et al., 1988), where it has revolutionizedour understanding of this disease (Akiyama et al., 2000). These ideashave been extended to other neurodegenerative diseases (Eikelenboom etal., 2002; Ishizawa and Dickson, 2001), to ischemic/toxic diseases(Gehrmann et al., 1995; Touzani et al., 1999), to tumor biology (Graeberet al., 2002) and even to normal brain development.

Neuroinflammation incorporates a wide spectrum of complex cellularresponses that include activation of microglia and astrocytes andinduction of cytokines, chemokines, complement proteins, acute phaseproteins, oxidative injury, and related molecular processes. Theseevents may have detrimental effects on neuronal function, leading toneuronal injury, further glial activation, and ultimatelyneurodegeneration.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with neuroinflammation.

H. Treatment of Renal Failure

Another aspect of the present disclosure concerns new methods andcompounds for the treatment and prevention of renal disease. See U.S.patent application Ser. No. 12/352,473, which is incorporated byreference herein in its entirety. Renal failure, resulting in inadequateclearance of metabolic waste products from the blood and abnormalconcentrations of electrolytes in the blood, is a significant medicalproblem throughout the world, especially in developed countries.Diabetes and hypertension are among the most important causes of chronicrenal failure (CKD), but it is also associated with other conditionssuch as lupus. Acute renal failure may arise from exposure to certaindrugs (e.g., acetaminophen) or toxic chemicals, or fromischemia-reperfusion injury associated with shock or surgical proceduressuch as transplantation, and may result in chronic renal failure. Inmany patients, renal failure advances to a stage in which the patientrequires regular dialysis or kidney transplantation to continue living.Both of these procedures are highly invasive and associated withsignificant side effects and quality of life issues. Although there areeffective treatments for some complications of renal failure, such ashyperparathyroidism and hyperphosphatemia, no available treatment hasbeen shown to halt or reverse the underlying progression of renalfailure. Thus, agents that can improve compromised renal function wouldrepresent a significant advance in the treatment of renal failure.

Inflammation contributes significantly to the pathology of CKD. There isalso a strong mechanistic link between oxidative stress and renaldysfunction. The NF-κB signaling pathway plays an important role in theprogression of CKD as NF-κB regulates the transcription of MCP-1, achemokine that is responsible for the recruitment ofmonocytes/macrophages resulting in an inflammatory response thatultimately injures the kidney (Wardle, 2001). The Keap1/Nrf2/ARE pathwaycontrols the transcription of several genes encoding antioxidantenzymes, including heme oxygenase-1 (HO-1). Ablation of the Nrf2 gene infemale mice results in the development of lupus-like glomerularnephritis (Yoh et al., 2001). Furthermore, several studies havedemonstrated that HO-1 expression is induced in response to renal damageand inflammation and that this enzyme and its products—bilirubin andcarbon monoxide—play a protective role in the kidney (Nath et al.,2006).

The glomerulus and the surrounding Bowman's capsule constitute the basicfunctional unit of the kidney. Glomerular filtration rate (GFR) is thestandard measure of renal function. Creatinine clearance is commonlyused to measure GFR. However, the level of serum creatinine is commonlyused as a surrogate measure of creatinine clearance. For instance,excessive levels of serum creatinine are generally accepted to indicateinadequate renal function and reductions in serum creatinine over timeare accepted as an indication of improved renal function. Normal levelsof creatinine in the blood are approximately 0.6 to 1.2 milligrams (mg)per deciliter (dl) in adult males and 0.5 to 1.1 milligrams perdeciliter in adult females.

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with renal failure.

I. Cardiovascular Disease

The compounds and methods of this invention may be used for treatingpatients with cardiovascular disease. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. Cardiovascular (CV) disease is among the most important causesof mortality worldwide, and is the leading cause of death in manydeveloped nations. The etiology of CV disease is complex, but themajority of causes are related to inadequate or completely disruptedsupply of blood to a critical organ or tissue. Frequently such acondition arises from the rupture of one or more atheroscleroticplaques, which leads to the formation of a thrombus that blocks bloodflow in a critical vessel. Such thrombosis is the principal cause ofheart attacks, in which one or more of the coronary arteries is blockedand blood flow to the heart itself is disrupted. The resulting ischemiais highly damaging to cardiac tissue, both from lack of oxygen duringthe ischemic event and from excessive formation of free radicals afterblood flow is restored (a phenomenon known as ischemia-reperfusioninjury). Similar damage occurs in the brain during a thrombotic stroke,when a cerebral artery or other major vessel is blocked by thrombosis.Hemorrhagic strokes, in contrast, involve rupture of a blood vessel andbleeding into the surrounding brain tissue. This creates oxidativestress in the immediate area of the hemorrhage, due to the presence oflarge amounts of free heme and other reactive species, and ischemia inother parts of the brain due to compromised blood flow. Subarachnoidhemorrhage, which is frequently accompanied by cerebral vasospasm, alsocauses ischemia/reperfusion injury in the brain.

Alternatively, atherosclerosis may be so extensive in critical bloodvessels that stenosis (narrowing of the arteries) develops and bloodflow to critical organs (including the heart) is chronicallyinsufficient. Such chronic ischemia can lead to end-organ damage of manykinds, including the cardiac hypertrophy associated with congestiveheart failure.

Atherosclerosis, the underlying defect leading to many forms ofcardiovascular disease, occurs when a physical defect or injury to thelining (endothelium) of an artery triggers an inflammatory responseinvolving the proliferation of vascular smooth muscle cells and theinfiltration of leukocytes into the affected area. Ultimately, acomplicated lesion known as an atherosclerotic plaque may form, composedof the above-mentioned cells combined with deposits ofcholesterol-bearing lipoproteins and other materials (e.g., Hansson etal., 2006).

Pharmaceutical treatments for cardiovascular disease include preventivetreatments, such as the use of drugs intended to lower blood pressure orcirculating levels of cholesterol and lipoproteins, as well astreatments designed to reduce the adherent tendencies of platelets andother blood cells (thereby reducing the rate of plaque progression andthe risk of thrombus formation). More recently, drugs such asstreptokinase and tissue plasminogen activator have been introduced andare used to dissolve the thrombus and restore blood flow. Surgicaltreatments include coronary artery bypass grafting to create analternative blood supply, balloon angioplasty to compress plaque tissueand increase the diameter of the arterial lumen, and carotidendarterectomy to remove plaque tissue in the carotid artery. Suchtreatments, especially balloon angioplasty, may be accompanied by theuse of stents, expandable mesh tubes designed to support the arterywalls in the affected area and keep the vessel open. Recently, the useof drug-eluting stents has become common in order to preventpost-surgical restenosis (renarrowing of the artery) in the affectedarea. These devices are wire stents coated with a biocompatible polymermatrix containing a drug that inhibits cell proliferation (e.g.,paclitaxel or rapamycin). The polymer allows a slow, localized releaseof the drug in the affected area with minimal exposure of non-targettissues. Despite the significant benefits offered by such treatments,mortality from cardiovascular disease remains high and significant unmetneeds in the treatment of cardiovascular disease remain.

As noted above, induction of HO-1 has been shown to be beneficial in avariety of models of cardiovascular disease, and low levels of HO-1expression have been clinically correlated with elevated risk of CVdisease. Compounds disclosed herein, therefore, may be used in treatingor preventing a variety of cardiovascular disorders including but notlimited to atherosclerosis, hypertension, myocardial infarction, chronicheart failure, stroke, subarachnoid hemorrhage, and restenosis.

J. Diabetes

Diabetes is a complex disease characterized by the body's failure toregulate circulating levels of glucose. See U.S. patent application Ser.No. 12/352,473, which is incorporated by reference herein in itsentirety. This failure may result from a lack of insulin, a peptidehormone that regulates the both the production and absorption of glucosein various tissues. Deficient insulin compromises the ability of muscle,fat, and other tissues to absorb glucose properly, leading tohyperglycemia (abnormally high levels of glucose in the blood). Mostcommonly, such insulin deficiency results from inadequate production inthe islet cells of the pancreas. In the majority of cases this arisesfrom autoimmune destruction of these cells, a condition known as type 1or juvenile-onset diabetes, but may also be due to physical trauma orsome other cause.

Diabetes may also arise when muscle and fat cells become less responsiveto insulin and do not absorb glucose properly, resulting inhyperglycemia. This phenomenon is known as insulin resistance, and theresulting condition is known as Type 2 diabetes. Type 2 diabetes, themost common type, is highly associated with obesity and hypertension.Obesity is associated with an inflammatory state of adipose tissue thatis thought to play a major role in the development of insulin resistance(e.g., Hotamisligil, 2006; Guilherme et al., 2008).

Diabetes is associated with damage to many tissues, largely becausehyperglycemia (and hypoglycemia, which can result from excessive orpoorly timed doses of insulin) is a significant source of oxidativestress. Chronic kidney failure, retinopathy, peripheral neuropathy,peripheral vasculitis, and the development of dermal ulcers that healslowly or not at all are among the common complications of diabetes.Because of their ability to protect against oxidative stress,particularly by the induction of HO-1 expression, compounds disclosedherein may be used in treatments for many complications of diabetes. Asnoted above (Cai et al., 2005), chronic inflammation and oxidativestress in the liver are suspected to be primary contributing factors inthe development of Type 2 diabetes. Furthermore, PPARγ agonists such asthiazolidinediones are capable of reducing insulin resistance and areknown to be effective treatments for Type 2 diabetes.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with neuroinflammation.

The effect of treatment of diabetes may be evaluated as follows. Boththe biological efficacy of the treatment modality as well as theclinical efficacy are evaluated, if possible. For example, diseasemanifests itself by increased blood sugar, the biological efficacy ofthe treatment therefore can be evaluated, for example, by observation ofreturn of the evaluated blood glucose towards normal. Measuring aclinical endpoint which can give an indication of b-cell regenerationafter, for example, a six-month period of time, can give an indicationof the clinical efficacy of the treatment regimen.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with diabetes.

K. Rheumatoid Arthritis

The compounds and methods of this invention may be used for treatingpatients with RA. Typically the first signs of rheumatoid arthritis (RA)appear in the synovial lining layer, with proliferation of synovialfibroblasts and their attachment to the articular surface at the jointmargin (Lipsky, 1998). Subsequently, macrophages, T cells and otherinflammatory cells are recruited into the joint, where they produce anumber of mediators, including the cytokines interleukin-1 (IL-1), whichcontributes to the chronic sequelae leading to bone and cartilagedestruction, and tumour necrosis factor (TNF-α), which plays a role ininflammation (Dinarello, 1998; Arend and Dayer, 1995; van den Berg,2001). The concentration of IL-1 in plasma is significantly higher inpatients with RA than in healthy individuals and, notably, plasma IL-1levels correlate with RA disease activity (Eastgate et al., 1988).Moreover, synovial fluid levels of IL-1 are correlated with variousradiographic and histologic features of RA (Kahle et al., 1992; Rooneyet al., 1990).

In normal joints, the effects of these and other proinflammatorycytokines are balanced by a variety of anti-inflammatory cytokines andregulatory factors (Burger and Dayer, 1995). The significance of thiscytokine balance is illustrated in juvenile RA patients, who havecyclical increases in fever throughout the day (Prieur et al., 1987).After each peak in fever, a factor that blocks the effects of IL-1 isfound in serum and urine. This factor has been isolated, cloned andidentified as IL-1 receptor antagonist (IL-1ra), a member of the IL-1gene family (Hannum et al., 1990). IL-1ra, as its name indicates, is anatural receptor antagonist that competes with IL-1 for binding to typeI IL-1 receptors and, as a result, blocks the effects of IL-1 (Arend etal., 1998). A 10- to 100-fold excess of IL-1ra may be needed to blockIL-1 effectively; however, synovial cells isolated from patients with RAdo not appear to produce enough IL-1ra to counteract the effects of IL-1(Firestein et al., 1994; Fujikawa et al., 1995).

L. Psoriatic Arthritis

Psoriasis is an inflammatory and proliferative skin disorder with aprevalence of 1.5-3%. Approximately 20% of patients with psoriasisdevelop a characteristic form of arthritis that has several patterns(Gladman, 1992; Jones et al., 1994; Gladman et al., 1995). Someindividuals present with joint symptoms first but in the majority, skinpsoriasis presents first. About one-third of patients have simultaneousexacerbations of their skin and joint disease (Gladman et al., 1987) andthere is a topographic relationship between nail and distalinterphalangeal joint disease (Jones et al., 1994; Wright, 1956).Although the inflammatory processes which link skin, nail and jointdisease remain elusive, an immune-mediated pathology is implicated.

Psoriatic arthritis (PsA) is a chronic inflammatory arthropathycharacterized by the association of arthritis and psoriasis and wasrecognized as a clinical entity distinct from rheumatoid arthritis (RA)in 1964 (Blumberg et al., 1964). Subsequent studies have revealed thatPsA shares a number of genetic, pathogenic and clinical features withother spondyloarthropathies (SpAs), a group of diseases that compriseankylosing spondylitis, reactive arthritis and enteropathic arthritis(Wright, 1979). The notion that PsA belongs to the SpA group hasrecently gained further support from imaging studies demonstratingwidespread enthesitis in the, including PsA but not RA (McGonagle etal., 1999; McGonagle et al., 1998). More specifically, enthesitis hasbeen postulated to be one of the earliest events occurring in the SpAs,leading to bone remodeling and ankylosis in the spine, as well as toarticular synovitis when the inflamed entheses are close to peripheraljoints. However, the link between enthesitis and the clinicalmanifestations in PsA remains largely unclear, as PsA can present withfairly heterogeneous patterns of joint involvement with variable degreesof severity (Marsal et al., 1999; Salvarani et al., 1998). Thus, otherfactors must be posited to account for the multifarious features of PsA,only a few of which (such as the expression of the HLA-B27 molecule,which is strongly associated with axial disease) have been identified.As a consequence, it remains difficult to map the disease manifestationsto specific pathogenic mechanisms, which means that the treatment ofthis condition remains largely empirical.

Family studies have suggested a genetic contribution to the developmentof PsA (Moll and Wright, 1973). Other chronic inflammatory forms ofarthritis, such as ankylosing spondylitis and rheumatoid arthritis, arethought to have a complex genetic basis. However, the genetic componentof PsA has been difficult to assess for several reasons. There is strongevidence for a genetic predisposition to psoriasis alone that may maskthe genetic factors that are important for the development of PsA.Although most would accept PsA as a distinct disease entity, at timesthere is a phenotypic overlap with rheumatoid arthritis and ankylosingspondylitis. Also, PsA itself is not a homogeneous condition and varioussubgroups have been proposed.

Increased amounts of TNF-α have been reported in both psoriatic skin(Ettehadi et al., 1994) and synovial fluid (Partsch et al., 1997).Recent trials have shown a positive benefit of anti-TNF treatment inboth PsA (Mease et al., 2000) and ankylosing spondylitis (Brandt et al.,2000).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with psoriatic arthritis.

M. Reactive Arthritis

In reactive arthritis (ReA) the mechanism of joint damage is unclear,but it is likely that cytokines play critical roles. A more prevalentTh1 profile high levels of interferon gamma (IFN-γ) and low levels ofinterleukin 4 (IL-4) has been reported (Lahesmaa et al., 1992; Schlaaket al., 1992; Simon et al., 1993; Schlaak et al., 1996; Kotake et al.,1999; Ribbens et al., 2000), but several studies have shown relativepredominance of IL-4 and IL-10 and relative lack of IFN-γ and tumournecrosis factor alpha (TNF-α) in the synovial membrane (Simon et al.,1994; Yin et al., 1999) and fluid (SF) (Yin et al., 1999; Yin et al.,1997) of reactive arthritis patients compared with rheumatoid arthritis(RA) patients. A lower level of TNF-α secretion in reactive arthritisthan in RA patients has also been reported after ex vivo stimulation ofperipheral blood mononuclear cells (PBMC) (Braun et al., 1999).

It has been argued that clearance of reactive arthritis-associatedbacteria requires the production of appropriate levels of IFN-γ andTNF-α, while IL-10 acts by suppressing these responses (Autenrieth etal., 1994; Sieper and Braun, 1995). IL-10 is a regulatory cytokine thatinhibits the synthesis of IL-12 and TNF-γ by activated macrophages (deWaal et al., 1991; Hart et al., 1995; Chomarat et al., 1995) and ofIFN-γ by T cells (Macatonia et al., 1993).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with reactive arthritis.

N. Enteropathic Arthritis

Typically enteropathic arthritis (EA) occurs in combination withinflammatory bowel diseases (IBD) such as Crohn's disease or ulcerativecolitis. It also can affect the spine and sacroiliac joints.Enteropathic arthritis involves the peripheral joints, usually in thelower extremities such as the knees or ankles. It commonly involves onlya few or a limited number of joints and may closely follow the bowelcondition. This occurs in approximately 11% of patients with ulcerativecolitis and 21% of those with Crohn's disease. The synovitis isgenerally self-limited and non-deforming.

Enteropathic arthropathies comprise a collection of rheumatologicconditions that share a link to GI pathology. These conditions includereactive (i.e., infection-related) arthritis due to bacteria (e.g.,Shigella, Salmonella, Campylobacter, Yersinia species, Clostridiumdifficile), parasites (e.g., Strongyloides stercoralis, Taenia saginata,Giardia lamblia, Ascaris lumbricoides, Cryptosporidium species), andspondyloarthropathies associated with inflammatory bowel disease (IBD).Other conditions and disorders include intestinal bypass (jejunoileal),arthritis, celiac disease, Whipple disease, and collagenous colitis.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with enteropathic arthritis.

O. Juvenile Rheumatoid Arthritis

Juvenile rheumatoid arthritis (JRA), a term for the most prevalent formof arthritis in children, is applied to a family of illnessescharacterized by chronic inflammation and hypertrophy of the synovialmembranes. The term overlaps, but is not completely synonymous, with thefamily of illnesses referred to as juvenile chronic arthritis and/orjuvenile idiopathic arthritis in Europe.

Both innate and adaptive immune systems use multiple cell types, a vastarray of cell surface and secreted proteins, and interconnected networksof positive and negative feedback (Lo et al., 1999). Furthermore, whileseparable in thought, the innate and adaptive wings of the immune systemare functionally intersected (Fearon and Locksley, 1996), and pathologicevents occurring at these intersecting points are likely to be highlyrelevant to our understanding of pathogenesis of adult and childhoodforms of chronic arthritis (Warrington, et al., 2001).

Polyarticular JRA is a distinct clinical subtype characterized byinflammation and synovial proliferation in multiple joints (four ormore), including the small joints of the hands (Jarvis, 2002). Thissubtype of JRA may be severe, because of both its multiple jointinvolvement and its capacity to progress rapidly over time. Althoughclinically distinct, polyarticular JRA is not homogeneous, and patientsvary in disease manifestations, age of onset, prognosis, and therapeuticresponse. These differences very likely reflect a spectrum of variationin the nature of the immune and inflammatory attack that can occur inthis disease (Jarvis, 1998).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with JRA.

P. Early Inflammatory Arthritis

The compounds and methods of this invention may be used for treatingpatients with early inflammatory arthritis. The clinical presentation ofdifferent inflammatory arthropathies is similar early in the course ofdisease. As a result, it is often difficult to distinguish patients whoare at risk of developing the severe and persistent synovitis that leadsto erosive joint damage from those whose arthritis is more self-limited.Such distinction is critical in order to target therapy appropriately,treating aggressively those with erosive disease and avoidingunnecessary toxicity in patients with more self-limited disease. Currentclinical criteria for diagnosing erosive arthropathies such asrheumatoid arthritis (RA) are less effective in early disease andtraditional markers of disease activity such as joint counts and acutephase response do not adequately identify patients likely to have pooroutcomes (Harrison et al., 1998). Parameters reflective of thepathologic events occurring in the synovium are most likely to be ofsignificant prognostic value.

Recent efforts to identify predictors of poor outcome in earlyinflammatory arthritis have identified the presence of RA specificautoantibodies, in particular antibodies towards citrullinated peptides,to be associated with erosive and persistent disease in earlyinflammatory arthritis cohorts. On the basis of this, a cyclicalcitrullinated peptide (CCP) has been developed to assist in theidentification of anti-CCP antibodies in patient sera. Using thisapproach, the presence of anti-CCP antibodies has been shown to bespecific and sensitive for RA, can distinguish RA from otherarthropathies, and can potentially predict persistent, erosive synovitisbefore these outcomes become clinically manifest. Importantly, anti-CCPantibodies are often detectable in sera many years prior to clinicalsymptoms suggesting that they may be reflective of subclinical immuneevents (Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003).

Q. Ankylosing Spondylitis

AS is a disease subset within a broader disease classification ofspondyloarthropathy. Patients affected with the various subsets ofspondyloarthropathy have disease etiologies that are often verydifferent, ranging from bacterial infections to inheritance. Yet, in allsubgroups, the end result of the disease process is axial arthritis.Despite the early clinically differences seen in the various patientpopulations, many of them end up nearly identical after a disease courseof ten-to-twenty years. Recent studies suggest the mean time to clinicaldiagnosis of ankylosing spondylitis from disease onset of disease is 7.5years (Khan, 1998). These same studies suggest that thespondyloarthropathies may have prevalence close to that of rheumatoidarthritis (Feldtkeller et al., 2003; Doran et al., 2003).

AS is a chronic systemic inflammatory rheumatic disorder of the axialskeleton with or without extraskeletal manifestations. Sacroiliac jointsand the spine are primarily affected, but hip and shoulder joints, andless commonly peripheral joints or certain extra-articular structuressuch as the eye, vasculature, nervous system, and gastrointestinalsystem may also be involved. Its etiology is not yet fully understood(Wordsworth, 1995; Calin and Taurog, 1998). It is strongly associatedwith the major histocompatibility class I (MHC I) HLA-B27 allele (Calinand Taurog, 1998). AS affects individuals in the prime of their life andis feared because of its potential to cause chronic pain andirreversible damage of tendons, ligaments, joints, and bones (Brewertonet al., 1973a; Brewerton et al., 1973b; Schlosstein et al., 1973). ASmay occur alone or in association with another form ofspondyloarthropathy such as reactive arthritis, psoriasis, psoriaticarthritis, enthesitis, ulcerative colitis, irritable bowel disease, orCrohn's disease, in which case it is classified as secondary AS.

Typically, the affected sites include the discovertebral, apophyseal,costovertebral, and costotransverse joints of the spine, and theparavertebral ligamentous structures. Inflammation of the entheses,which are sites of musculotendinous and ligamentous attachment to bones,is also prominent in this disease (Calin and Taurog, 1998). The site ofenthesitis is known to be infiltrated by plasma cells, lymphocytes, andpolymorphonuclear cells. The inflammatory process frequently results ingradual fibrous and bony ankylosis, (Ball, 1971; Khan, 1990).

Delayed diagnosis is common because symptoms are often attributed tomore common back problems. A dramatic loss of flexibility in the lumbarspine is an early sign of AS. Other common symptoms include chronic painand stiffness in the lower back which usually starts where the lowerspine is joined to the pelvis, or hip. Although most symptoms begin inthe lumbar and sacroiliac areas, they may involve the neck and upperback as well. Arthritis may also occur in the shoulder, hips and feet.Some patients have eye inflammation, and more severe cases must beobserved for heart valve involvement.

The most frequent presentation is back pain, but disease can beginatypically in peripheral joints, especially in children and women, andrarely with acute iritis (anterior uveitis). Additional early symptomsand signs are diminished chest expansion from diffuse costovertebralinvolvement, low-grade fever, fatigue, anorexia, weight loss, andanemia. Recurrent back pain—often nocturnal and of varying intensity—isan eventual complaint, as is morning stiffness typically relieved byactivity. A flexed or bent-over posture eases back pain and paraspinalmuscle spasm; thus, some degree of kyphosis is common in untreatedpatients.

Systemic manifestations occur in ⅓ of patients. Recurrent, usuallyself-limited, acute iritis (anterior uveitis) rarely is protracted andsevere enough to impair vision. Neurologic signs can occasionally resultfrom compression radiculitis or sciatica, vertebral fracture orsubluxation, and cauda equina syndrome (which consists of impotence,nocturnal urinary incontinence, diminished bladder and rectal sensation,and absence of ankle jerks). Cardiovascular manifestations can includeaortic insufficiency, angina, pericarditis, and ECG conductionabnormalities. A rare pulmonary finding is upper lobe fibrosis,occasionally with cavitation that may be mistaken for TB and can becomplicated by infection with Aspergillus.

AS is characterized by mild or moderate flares of active spondylitisalternating with periods of almost or totally inactive inflammation.Proper treatment in most patients results in minimal or no disabilityand in full, productive lives despite back stiffness. Occasionally, thecourse is severe and progressive, resulting in pronounced incapacitatingdeformities. The prognosis is bleak for patients with refractory iritisand for the rare patient with secondary amyloidosis.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with ankylosing spondylitis.

R. Ulcerative Colitis

Ulcerative colitis is a disease that causes inflammation and sores,called ulcers, in the lining of the large intestine. The inflammationusually occurs in the rectum and lower part of the colon, but it mayaffect the entire colon. Ulcerative colitis rarely affects the smallintestine except for the end section, called the terminal ileum.Ulcerative colitis may also be called colitis or proctitis. Theinflammation makes the colon empty frequently, causing diarrhea. Ulcersform in places where the inflammation has killed the cells lining thecolon; the ulcers bleed and produce pus.

Ulcerative colitis is an inflammatory bowel disease (IBD), the generalname for diseases that cause inflammation in the small intestine andcolon. Ulcerative colitis can be difficult to diagnose because itssymptoms are similar to other intestinal disorders and to another typeof IBD, Crohn's disease. Crohn's disease differs from ulcerative colitisbecause it causes inflammation deeper within the intestinal wall. Also,Crohn's disease usually occurs in the small intestine, although it canalso occur in the mouth, esophagus, stomach, duodenum, large intestine,appendix, and anus.

Ulcerative colitis may occur in people of any age, but most often itstarts between ages 15 and 30, or less frequently between ages 50 and70. Children and adolescents sometimes develop the disease. Ulcerativecolitis affects men and women equally and appears to run in somefamilies. Theories about what causes ulcerative colitis abound, but nonehave been proven. The most popular theory is that the body's immunesystem reacts to a virus or a bacterium by causing ongoing inflammationin the intestinal wall. People with ulcerative colitis haveabnormalities of the immune system, but doctors do not know whetherthese abnormalities are a cause or a result of the disease. Ulcerativecolitis is not caused by emotional distress or sensitivity to certainfoods or food products, but these factors may trigger symptoms in somepeople.

The most common symptoms of ulcerative colitis are abdominal pain andbloody diarrhea. Patients also may experience fatigue, weight loss, lossof appetite, rectal bleeding, and loss of body fluids and nutrients.About half of patients have mild symptoms. Others suffer frequent fever,bloody diarrhea, nausea, and severe abdominal cramps. Ulcerative colitismay also cause problems such as arthritis, inflammation of the eye,liver disease (hepatitis, cirrhosis, and primary sclerosingcholangitis), osteoporosis, skin rashes, and anemia. No one knows forsure why problems occur outside the colon. Scientists think thesecomplications may occur when the immune system triggers inflammation inother parts of the body. Some of these problems go away when the colitisis treated.

A thorough physical exam and a series of tests may be required todiagnose ulcerative colitis. Blood tests may be done to check foranemia, which could indicate bleeding in the colon or rectum. Bloodtests may also uncover a high white blood cell count, which is a sign ofinflammation somewhere in the body. By testing a stool sample, thedoctor can detect bleeding or infection in the colon or rectum. Thedoctor may do a colonoscopy or sigmoidoscopy. For either test, thedoctor inserts an endoscope—a long, flexible, lighted tube connected toa computer and TV monitor—into the anus to see the inside of the colonand rectum. The doctor will be able to see any inflammation, bleeding,or ulcers on the colon wall. During the exam, the doctor may do abiopsy, which involves taking a sample of tissue from the lining of thecolon to view with a microscope. A barium enema x ray of the colon mayalso be required. This procedure involves filling the colon with barium,a chalky white solution. The barium shows up white on x-ray film,allowing the doctor a clear view of the colon, including any ulcers orother abnormalities that might be there.

Treatment for ulcerative colitis depends on the seriousness of thedisease. Most people are treated with medication. In severe cases, apatient may need surgery to remove the diseased colon. Surgery is theonly cure for ulcerative colitis. Some people whose symptoms aretriggered by certain foods are able to control the symptoms by avoidingfoods that upset their intestines, like highly seasoned foods, rawfruits and vegetables, or milk sugar (lactose). Each person mayexperience ulcerative colitis differently, so treatment is adjusted foreach individual. Emotional and psychological support is important. Somepeople have remissions—periods when the symptoms go away—that last formonths or even years. However, most patients' symptoms eventuallyreturn. This changing pattern of the disease means one cannot alwaystell when a treatment has helped. Some people with ulcerative colitismay need medical care for some time, with regular doctor visits tomonitor the condition.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with ulcerative colitis.

S. Crohn's Disease

Another disorder for which immunosuppression has been tried is Crohn'sdisease. Crohn's disease symptoms include intestinal inflammation andthe development of intestinal stenosis and fistulas; neuropathy oftenaccompanies these symptoms. Anti-inflammatory drugs, such as5-aminosalicylates (e.g., mesalamine) or corticosteroids, are typicallyprescribed, but are not always effective (reviewed in Botoman et al.,1998). Immunosuppression with cyclosporine is sometimes beneficial forpatients resistant to or intolerant of corticosteroids (Brynskov et al.,1989).

Efforts to develop diagnostic and treatment tools against Crohn'sdisease have focused on the central role of cytokines (Schreiber, 1998;van Hogezand and Verspaget, 1998). Cytokines are small secreted proteinsor factors (5 to 20 kD) that have specific effects on cell-to-cellinteractions, intercellular communication, or the behavior of othercells. Cytokines are produced by lymphocytes, especially T_(H)1 andT_(H)2 lymphocytes, monocytes, intestinal macrophages, granulocytes,epithelial cells, and fibroblasts (reviewed in Rogler and. Andus, 1998;Galley and Webster, 1996). Some cytokines are pro-inflammatory (e.g.,TNF-α, IL-1(α and β), IL-6, IL-8, IL-12, or leukemia inhibitory factor[LIF]); others are anti-inflammatory (e.g., IL-1 receptor antagonist,IL-4, IL-10, IL-11, and TGF-β). However, there may be overlap andfunctional redundancy in their effects under certain inflammatoryconditions.

In active cases of Crohn's disease, elevated concentrations of TNF-α andIL-6 are secreted into the blood circulation, and TNF-α, IL-1, IL-6, andIL-8 are produced in excess locally by mucosal cells (id.; Funakoshi etal., 1998). These cytokines can have far-ranging effects onphysiological systems including bone development, hematopoiesis, andliver, thyroid, and neuropsychiatric function. Also, an imbalance of theIL-1β/IL-1ra ratio, in favor of pro-inflammatory IL-1β, has beenobserved in patients with Crohn's disease (Rogler and Andus, 1998; Saikiet al., 1998; Dionne et al., 1998; but see Kuboyama, 1998). One studysuggested that cytokine profiles in stool samples could be a usefuldiagnostic tool for Crohn's disease (Saiki et al., 1998).

Treatments that have been proposed for Crohn's disease include the useof various cytokine antagonists (e.g., IL-1ra), inhibitors (e.g., ofIL-1β converting enzyme and antioxidants) and anti-cytokine antibodies(Rogler and Andus, 1998; van Hogezand and Verspaget, 1998; Reimund etal., 1998; Lugering et al., 1998; McAlindon et al., 1998). Inparticular, monoclonal antibodies against TNF-α have been tried withsome success in the treatment of Crohn's disease (Targan et al., 1997;Stack et al., 1997; van Dullemen et al., 1995). These compounds may beused in combination therapy with compounds of the present disclosure.

Another approach to the treatment of Crohn's disease has focused on atleast partially eradicating the bacterial community that may betriggering the inflammatory response and replacing it with anon-pathogenic community. For example, U.S. Pat. No. 5,599,795 disclosesa method for the prevention and treatment of Crohn's disease in humanpatients. Their method was directed to sterilizing the intestinal tractwith at least one antibiotic and at least one anti-fungal agent to killoff the existing flora and replacing them with different, select,well-characterized bacteria taken from normal humans. Borody taught amethod of treating Crohn's disease by at least partial removal of theexisting intestinal microflora by lavage and replacement with a newbacterial community introduced by fecal inoculum from a disease-screenedhuman donor or by a composition comprising Bacteroides and Escherichiacoli species. (U.S. Pat. No. 5,443,826).

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with Crohn's disease.

T. Systemic Lupus Erythematosus

There has also been no known cause for autoimmune diseases such assystemic lupus erythematosus. Systemic lupus erythematosus (SLE) is anautoimmune rheumatic disease characterized by deposition in tissues ofautoantibodies and immune complexes leading to tissue injury (Kotzin,1996). In contrast to autoimmune diseases such as MS and type 1 diabetesmellitus, SLE potentially involves multiple organ systems directly, andits clinical manifestations are diverse and variable (reviewed by Kotzinand O'Dell, 1995). For example, some patients may demonstrate primarilyskin rash and joint pain, show spontaneous remissions, and requirelittle medication. At the other end of the spectrum are patients whodemonstrate severe and progressive kidney involvement that requirestherapy with high doses of steroids and cytotoxic drugs such ascyclophosphamide (Kotzin, 1996).

The serological hallmark of SLE, and the primary diagnostic testavailable, is elevated serum levels of IgG antibodies to constituents ofthe cell nucleus, such as double-stranded DNA (dsDNA), single-strandedDNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNAantibodies play a major role in the development of lupusglomerulonephritis (G N) (Hahn and Tsao, 1993; Ohnishi et al., 1994).Glomerulonephritis is a serious condition in which the capillary wallsof the kidney's blood purifying glomeruli become thickened by accretionson the epithelial side of glomerular basement membranes. The disease isoften chronic and progressive and may lead to eventual renal failure.

Based on experimental results obtained, including those presented inthis application, the compounds and methods of this invention may beused for treating patients with SLE.

U. Irritable Bowel Syndrome

The compounds and methods of this invention may be used for treatingpatients with Irritable bowel syndrome (IBS). IBS is a functionaldisorder characterized by abdominal pain and altered bowel habits. Thissyndrome may begin in young adulthood and can be associated withsignificant disability. This syndrome is not a homogeneous disorder.Rather, subtypes of IBS have been described on the basis of thepredominant symptom—diarrhea, constipation, or pain. In the absence of“alarm” symptoms, such as fever, weight loss, and gastrointestinalbleeding, a limited workup is needed. Once a diagnosis of IBS is made,an integrated treatment approach can effectively reduce the severity ofsymptoms. IBS is a common disorder, although its prevalence rates havevaried. In general, IBS affects about 15% of US adults and occurs aboutthree times more often in women than in men (Jailwala et al., 2000).

IBS accounts for between 2.4 million and 3.5 million visits tophysicians each year. It not only is the most common condition seen bygastroenterologists but also is one of the most common gastrointestinalconditions seen by primary care physicians (Everhart et al., 1991;Sandler, 1990).

IBS is also a costly disorder. Compared with persons who do not havebowel symptoms, persons with IBS miss three times as many workdays andare more likely to report being too sick to work (Drossman et al., 1993;Drossman et al., 1997). Moreover, those with IBS incur hundreds ofdollars more in medical charges than persons without bowel disorders(Talley et al., 1995).

No specific abnormality accounts for the exacerbations and remissions ofabdominal pain and altered bowel habits experienced by patients withIBS. The evolving theory of IBS suggests dysregulation at multiplelevels of the brain-gut axis. Dysmotility, visceral hypersensitivity,abnormal modulation of the central nervous system (CNS), and infectionhave all been implicated. In addition, psychosocial factors play animportant modifying role. Abnormal intestinal motility has long beenconsidered a factor in the pathogenesis of IBS. Transit time through thesmall intestine after a meal has been shown to be shorter in patientswith diarrhea-predominant IBS than in patients who have theconstipation-predominant or pain-predominant subtype (Cann et al.,1983).

In studies of the small intestine during fasting, the presence of bothdiscrete, clustered contractions and prolonged, propagated contractionshas been reported in patients with IBS (Kellow and Phillips, 1987). Theyalso experience pain with irregular contractions more often than healthypersons (Kellow and Phillips, 1987; Horwitz and Fisher, 2001)

These motility findings do not account for the entire symptom complex inpatients with IBS; in fact, most of these patients do not havedemonstrable abnormalities (Rothstein, 2000). Patients with IBS haveincreased sensitivity to visceral pain. Studies involving balloondistention of the rectosigmoid colon have shown that patients with IBSexperience pain and bloating at pressures and volumes much lower thancontrol subjects (Whitehead et al., 1990). These patients maintainnormal perception of somatic stimuli.

Multiple theories have been proposed to explain this phenomenon. Forexample, receptors in the viscera may have increased sensitivity inresponse to distention or intraluminal contents. Neurons in the dorsalhorn of the spinal cord may have increased excitability. In addition,alteration in CNS processing of sensations may be involved (Drossman etal., 1997). Functional magnetic resonance imaging studies have recentlyshown that compared with control subjects, patients with IBS haveincreased activation of the anterior cingulate cortex, an important paincenter, in response to a painful rectal stimulus (Mertz et al., 2000).

Increasingly, evidence suggests a relationship between infectiousenteritis and subsequent development of IBS. Inflammatory cytokines mayplay a role. In a survey of patients with a history of confirmedbacterial gastroenteritis (Neal et al., 1997), 25% reported persistentalteration of bowel habits. Persistence of symptoms may be due topsychological stress at the time of acute infection (Gwee et al., 1999).

Recent data suggest that bacterial overgrowth in the small intestine mayhave a role in IBS symptoms. In one study (Pimentel et al., 2000), 157(78%) of 202 IBS patients referred for hydrogen breath testing had testfindings that were positive for bacterial overgrowth. Of the 47 subjectswho had follow-up testing, 25 (53%) reported improvement in symptoms(i.e., abdominal pain and diarrhea) with antibiotic treatment.

IBS may present with a range of symptoms. However, abdominal pain andaltered bowel habits remain the primary features. Abdominal discomfortis often described as crampy in nature and located in the left lowerquadrant, although the severity and location can differ greatly.Patients may report diarrhea, constipation, or alternating episodes ofdiarrhea and constipation. Diarrheal symptoms are typically described assmall-volume, loose stools, and stool is sometimes accompanied by mucusdischarge. Patients also may report bloating, fecal urgency, incompleteevacuation, and abdominal distention. Upper gastrointestinal symptoms,such as gastroesophageal reflux, dyspepsia, or nausea, may also bepresent (Lynn and Friedman, 1993).

Persistence of symptoms is not an indication for further testing; it isa characteristic of IBS and is itself an expected symptom of thesyndrome. More extensive diagnostic evaluation is indicated in patientswhose symptoms are worsening or changing. Indications for furthertesting also include presence of alarm symptoms, onset of symptoms afterage 50, and a family history of colon cancer. Tests may includecolonoscopy, computed tomography of the abdomen and pelvis, and bariumstudies of the small or large intestine.

V. Sjögren's Syndrome

The compounds and methods of this invention may be used for treatingpatients with SS. Primary Sjögren's syndrome (SS) is a chronic, slowlyprogressive, systemic autoimmune disease, which affects predominantlymiddle-aged women (female-to-male ratio 9:1), although it can be seen inall ages including childhood (Jonsson et al., 2002). It is characterizedby lymphocytic infiltration and destruction of the exocrine glands,which are infiltrated by mononuclear cells including CD4+, CD8+lymphocytes and B-cells (Jonsson et al., 2002). In addition,extraglandular (systemic) manifestations are seen in one-third ofpatients (Jonsson et al., 2001).

The glandular lymphocytic infiltration is a progressive feature (Jonssonet al., 1993), which, when extensive, may replace large portions of theorgans. Interestingly, the glandular infiltrates in some patientsclosely resemble ectopic lymphoid microstructures in the salivary glands(denoted as ectopic germinal centers) (Salomonsson et al., 2002; Xanthouet al., 2001). In SS, ectopic GCs are defined as T and B cell aggregatesof proliferating cells with a network of follicular dendritic cells andactivated endothelial cells. These GC-like structures formed within thetarget tissue also portray functional properties with production ofautoantibodies (anti-Ro/SSA and anti-La/SSB) (Salomonsson and Jonsson,2003).

In other systemic autoimmune diseases, such as RA, factors critical forectopic GCs have been identified. Rheumatoid synovial tissues with GCswere shown to produce chemokines CXCL13, CCL21 and lymphotoxin (LT)-β(detected on follicular center and mantle zone B cells). Multivariateregression analysis of these analytes identified CXCL13 and LT-β as thesolitary cytokines predicting GCs in rheumatoid synovitis (Weyand andGoronzy, 2003). Recently CXCL13 and CXCR5 in salivary glands has beenshown to play an essential role in the inflammatory process byrecruiting B and T cells, therefore contributing to lymphoid neogenesisand ectopic GC formation in SS (Salomonsson et al., 2002).

W. Psoriasis

The compounds and methods of this invention may be used for treatingpatients with psoriasis. Psoriasis is a chronic skin disease of scalingand inflammation that affects 2 to 2.6 percent of the United Statespopulation, or between 5.8 and 7.5 million people. Although the diseaseoccurs in all age groups, it primarily affects adults. It appears aboutequally in males and females. Psoriasis occurs when skin cells quicklyrise from their origin below the surface of the skin and pile up on thesurface before they have a chance to mature. Usually this movement (alsocalled turnover) takes about a month, but in psoriasis it may occur inonly a few days. In its typical form, psoriasis results in patches ofthick, red (inflamed) skin covered with silvery scales. These patches,which are sometimes referred to as plaques, usually itch or feel sore.They most often occur on the elbows, knees, other parts of the legs,scalp, lower back, face, palms, and soles of the feet, but they canoccur on skin anywhere on the body. The disease may also affect thefingernails, the toenails, and the soft tissues of the genitals andinside the mouth. While it is not unusual for the skin around affectedjoints to crack, approximately 1 million people with psoriasisexperience joint inflammation that produces symptoms of arthritis. Thiscondition is called psoriatic arthritis.

Psoriasis is a skin disorder driven by the immune system, especiallyinvolving a type of white blood cell called a T cell. Normally, T cellshelp protect the body against infection and disease. In the case ofpsoriasis, T cells are put into action by mistake and become so activethat they trigger other immune responses, which lead to inflammation andto rapid turnover of skin cells. In about one-third of the cases, thereis a family history of psoriasis. Researchers have studied a largenumber of families affected by psoriasis and identified genes linked tothe disease. People with psoriasis may notice that there are times whentheir skin worsens, then improves. Conditions that may cause flareupsinclude infections, stress, and changes in climate that dry the skin.Also, certain medicines, including lithium and beta blockers, which areprescribed for high blood pressure, may trigger an outbreak or worsenthe disease.

X. Infectious Diseases

Compounds of the present disclosure may be useful in the treatment ofinfectious diseases, including viral and bacterial infections. As notedabove, such infections may be associated with severe localized orsystemic inflammatory responses. For example, influenza may cause severeinflammation of the lung and bacterial infection can cause the systemichyperinflammatory response, including the excessive production ofmultiple inflammatory cytokines, that is the hallmark of sepsis. Inaddition, compounds disclosed herein may be useful in directlyinhibiting the replication of viral pathogens. Previous studies havedemonstrated that related compounds such as CDDO can inhibit thereplication of HIV in macrophages (Vazquez et al., 2005). Other studieshave indicated that inhibition of NF-kappa B signaling may inhibitinfluenza virus replication, and that cyclopentenone prostaglandins mayinhibit viral replication (e.g., Mazur et al., 2007; Pica et al., 2000).

V. Pharmaceutical Formulations and Routes of Administration

The compounds of the present disclosure may be administered by a varietyof methods, e.g., orally or by injection (e.g. subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, the active compounds may be coated in a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

Active compounds are administered at a therapeutically effective dosagesufficient to treat a condition associated with a condition in apatient. A “therapeutically effective amount” preferably reduces theamount of symptoms of the condition in the infected patient by at leastabout 20%, more preferably by at least about 40%, even more preferablyby at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. For example, the efficacy of a compoundcan be evaluated in an animal model system that may be predictive ofefficacy in treating the disease in humans, such as the model systemsshown in the examples and drawings.

The actual dosage amount of a compound of the present disclosure orcomposition comprising a compound of the present disclosure administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

An effective amount typically will vary from about 0.001 mg/kg to about1000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 100mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about 250 mg/kg, fromabout 10.0 mg/kg to about 150 mg/kg in one or more dose administrationsdaily, for one or several days (depending of course of the mode ofadministration and the factors discussed above). Other suitable doseranges include 1 mg to 10000 mg per day, 100 mg to 10000 mg per day, 500mg to 10000 mg per day, and 500 mg to 1000 mg per day. In someparticular embodiments, the amount is less than 10,000 mg per day with arange of 750 mg to 9000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day or less than 10 mg/kg/day. It mayalternatively be in the range of 1 mg/kg/day to 200 mg/kg/day. Forexample, regarding treatment of diabetic patients, the unit dosage maybe an amount that reduces blood glucose by at least 40% as compared toan untreated subject. In another embodiment, the unit dosage is anamount that reduces blood glucose to a level that is ±10% of the bloodglucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1micro-gram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentdisclosure may comprise, for example, at least about 0.1% of a compoundof the present disclosure. In other embodiments, the compound of thepresent disclosure may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may taken orallyand that the timing of which is or is not dependent upon food intake.Thus, for example, the agent can be taken every morning and/or everyevening, regardless of when the subject has eaten or will eat.

VI. Combination Therapy

In addition to being used as a monotherapy, the compounds of the presentdisclosure may also find use in combination therapies. Effectivecombination therapy may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, at the same time, wherein onecomposition includes a compound of the present disclosure, and the otherincludes the second agent(s). Alternatively, the therapy may precede orfollow the other agent treatment by intervals ranging from minutes tomonths.

Various combinations may be employed, such as when a compound of thepresent disclosure is “A” and “B” represents a secondary agent,non-limiting examples of which are described below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/BB/B/B/A  B/B/A/B  A/A/B/B  A/B/A/B  A/B/B/A  B/B/A/AB/A/B/A  B/A/A/B  A/A/A/B  B/A/A/A  A/B/A/A  A/A/B/A

Administration of the compounds of the present disclosure to a patientwill follow general protocols for the administration of pharmaceuticals,taking into account the toxicity, if any, of the drug. It is expectedthat the treatment cycles would be repeated as necessary.

Beta interferons may be suitable secondary agents. These are medicationsderived from human cytokines which help regulate the immune system. Theyinclude interferon β-1b and interferon β-1a. Betaseron has been approvedby the FDA for relapsing forms of secondary progressive MS. Furthermore,the FDA has approved the use of several β-interferons as treatments forpeople who have experienced a single attack that suggests multiplesclerosis, and who may be at risk of future attacks and developingdefinite MS. For example, risk of MS may be suggested when an MRI scanof the brain shows lesions that predict a high risk of conversion todefinite MS.

Glatiramer acetate is a further example of a secondary agent that may beused in a combination treatment. Glatiramer is presently used to treatrelapsing remitting MS. It is made of four amino acids that are found inmyelin. This drug is reported to stimulate T cells in the body's immunesystem to change from harmful, pro-inflammatory agents to beneficial,anti-inflammatory agents that work to reduce inflammation at lesionsites.

Another potential secondary agent is mitoxantrone, a chemotherapy drugused for many cancers. This drug is also FDA-approved for treatment ofaggressive forms of relapsing remitting MS, as well as certain forms ofprogressive MS. It is given intravenously, typically every three months.This medication is effective, but is limited by cardiac toxicity.Novantrone has been approved by the FDA for secondary progressive,progressive-relapsing, and worsening relapsing-remitting MS.

Another potential secondary agent is natalizumab. In general,natalizumab works by blocking the attachment of immune cells to brainblood vessels, which is a necessary step for immune cells to cross intothe brain, thus reducing the immune cells' inflammatory action on brainneurons. Natalizumab has been shown to significantly reduce thefrequency of attacks in people with relapsing MS.

In the case of relapsing remitting MS, patients may be given intravenouscorticosteroids, such as methylprednisolone, as a secondary agent, toend the attack sooner and leave fewer lasting deficits.

Other common drugs for MS that may be used in combination with compoundsof the present disclosure include immunosuppressive drugs such asazathioprine, cladribine, and cyclophosphamide.

It is contemplated that other anti-inflammatory agents may be used inconjunction with the treatments of the current invention. Other COXinhibitors may be used, including arylcarboxylic acids (salicylic acid,acetylsalicylic acid, diflunisal, choline magnesium trisalicylate,salicylate, benorylate, flufenamic acid, mefenamic acid, meclofenamicacid and triflumic acid), arylalkanoic acids (diclofenac, fenclofenac,alclofenac, fentiazac, ibuprofen, flurbiprofen, ketoprofen, naproxen,fenoprofen, fenbufen, suprofen, indoprofen, tiaprofenic acid,benoxaprofen, pirprofen, tolmetin, zomepirac, clopinac, indomethacin andsulindac) and enolic acids (phenylbutazone, oxyphenbutazone,azapropazone, feprazone, piroxicam, and isoxicam. See, e.g., U.S. Pat.No. 6,025,395.

Histamine H2 receptor blocking agents may also be used in conjunctionwith the compounds of the current invention, including cimetidine,ranitidine, famotidine and nizatidine.

Treatment with acetylcholinesterase inhibitors such as tacrine,donepizil, metrifonate and rivastigmine for the treatment of Alzheimer'sand other disease in conjunction with the compounds of the presentdisclosure is contemplated. Other acetylcholinesterase inhibitors may bedeveloped which may be used once approved include rivastigmine andmetrifonate. Acetylcholinesterase inhibitors increase the amount ofneurotransmitter acetylcholine at the nerve terminal by decreasing itsbreakdown by the enzyme cholinesterase.

MAO-B inhibitors such as selegilene may be used in conjunction with thecompounds of the current invention. Selegilene is used for Parkinson'sdisease and irreversibly inhibits monoamine oxidase type B (MAO-B).Monoamine oxidase is an enzyme that inactivates the monoamineneurotransmitters norepinephrine, serotonin and dopamine.

Dietary and nutritional supplements with reported benefits for treatmentor prevention of Parkinson's, Alzheimer's, multiple sclerosis,amyotrophic lateral sclerosis, rheumatoid arthritis, inflammatory boweldisease, and all other diseases whose pathogenesis is believed toinvolve excessive production of either nitric oxide (NO) orprostaglandins, such as acetyl-L-carnitine, octacosanol, eveningprimrose oil, vitamin B6, tyrosine, phenylalanine, vitamin C, L-dopa, ora combination of several antioxidants may be used in conjunction withthe compounds of the current invention.

For the treatment or prevention of cancer, compounds disclosed hereinmay be combined with one or more of the following: radiation,chemotherapy agents (e.g., cytotoxic agents such as anthracyclines,vincristine, vinblastin, microtubule-targeting agents such as paclitaxeland docetaxel, 5-FU and related agents, cisplatin and otherplatinum-containing compounds, irinotecan and topotecan, gemcitabine,temozolomide, etc.), targeted therapies (e.g., imatinib, bortezomib,bevacizumab, rituximab), or vaccine therapies designed to promote anenhanced immune response targeting cancer cells.

For the treatment or prevention of autoimmune disease, compoundsdisclosed herein may be combined with one or more of the following:corticosteroids, methotrexate, anti-TNF antibodies, other TNF-targetingprotein therapies, and NSAIDs. For the treatment of prevention ofcardiovascular diseases, compounds disclosed herein may be combined withantithrombotic therapies, anticholesterol therapies such as statins(e.g., atorvastatin), and surgical interventions such as stenting orcoronary artery bypass grafting. For the treatment of osteoporosis,compounds disclosed herein may be combined with antiresorptive agentssuch as bisphosphonates or anabolic therapies such as teriparatide orparathyroid hormone. For the treatment of neuropsychiatric conditions,compounds disclosed herein may be combined with antidepressants (e.g.,imipramine or SSRIs such as fluoxetine), antipsychotic agents (e.g.,olanzapine, sertindole, risperidone), mood stabilizers (e.g., lithium,valproate semisodium), or other standard agents such as anxiolyticagents. For the treatment of neurological disorders, compounds disclosedherein may be combined with anticonvulsant agents (e.g., valproatesemisodium, gabapentin, phenyloin, carbamazepine, and topiramate),antithrombotic agents (e.g., tissue plasminogen activator), oranalgesics (e.g., opioids, sodium channel blockers, and otherantinociceptive agents).

VII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Methods and Materials

UV spectra were measured on Shimadzu UV1201 UV-VIS spectrophotometer.All NMR spectra were recorded on a Fourier transform spectrometer. Thetwo instruments used were either 500 MHz for ¹H and 125 MHz for ¹³C or300 MHz for ¹H and 75 MHz for ¹³C. The chemical shifts are reported in δ(ppm) using the δ 7.27 signal of CHCl₃ (¹H NMR) and the δ 77.23 signalof CDCl₃ (¹³C NMR) as internal standards for deuterated chlorform, the δ2.05 signal of CD₃COCHD₂ (¹H NMR) and the δ 29.92 signal of CD₃COCD₃(¹³C NMR) as internal standards for acetone-d₆. Coupling constants arereported in hertz (Hz) and the apparent multiplicity is described ass=singlet, m=multiplet, d=doublet, and t=triplet.

Low resolution and high resolution mass spectroscopy data were obtainedby the ESI+ and EI.

Precoated TLC plates with silica gel 60 F254 were used for TLC andpreparative TLC.

Flash column chromatography was performed with silica gel (230-400mesh). All experiments were performed under a nitrogen atmosphere unlessstated differently.

Anhydrous tetraydrofuran and dichloromethane were prepared by a solventpurification system. All solvents (analytical grade) including anhydroussolvents and reagents were used as received. All references to “water”correspond to reverse osmosis deionized (RODI) water. All references to“brine” refer to a saturated aqueous sodium chloride solution.

The term “in vacuo” refers to solvent removal by rotary evaporationfollowed by a lower pressure environment (≦0.2 Torr).

For the iNOS assay, RAW cells were treated with various concentrationsof compounds and interferon-γ (10 ng/mL) for 24 h. Supernatants wereanalyzed for nitric oxide (NO) by the Griess reaction. IC₅₀ values arean average of two separate experiments.

In vitro IKKβ kinase assay using commercially available HTScan® IKKβKinase Assay Kit. The protocol is shown as follows: 1. One hundred μL of10 mM ATP is added to 1.25 mL of 6 μM IκB-α (Ser32) biotinylated peptide(substrate peptide). The mixture was diluted with deionized H₂O to 2.5mL to make ATP/substrate cocktail ([ATP]=400 μM, [substrate]=3 μM). 2.IKKβ Kinase (recombinant, human) (enzyme, 5 μg) is transferred from −80°C. to ice. The enzyme is allowed to thaw on ice. 3. The enzyme ismicrocentrifuged briefly at 4° C. to bring liquid to the bottom of thevial. The enzyme is returned immediately to ice. 4. One mL of kinasebuffer [1 mL of Kinase Buffer: 250 mM Tris-HCl pH 7.5, 100 mM MgCl₂, 1mM Na₃VO₄, 50 mM β-glycerophosphate] is added to 1.5 mL of deionized H₂Oto make 2.5 mL of reaction buffer. 5. One and quarter mL of the reactionbuffer is transferred to the enzyme tube to make reaction cocktail([enzyme])=4 ng/μL in the reaction cocktail). 6. Twelve and a half μL ofthe reaction cocktail is added to 12.5 μL/well of solution of newligands at each concentration and the mixture is incubated for 5 minutesat room temperature. 7. Twentyfive μL of the ATP/substrate cocktail isadded to 25 μL/well preincubated reaction cocktail/compound. [At thisstage, Final Assay Conditions for a 50 μL Reaction are 25 mM Tris-HCl(pH 7.5), 10 mM MgCl₂, 5 mM β-glycerophosphate, 0.1 mM Na₃VO₄, 200 μMATP, 1.5 μM peptide, 50 ng IKKβ Kinase] 8. The reaction plate isincubated at room temperature for 30 minutes. 9. Fifty μL/well StopBuffer (50 mM EDTA, pH 8) is added to stop the reaction. 10. Twenty-fiveμL of each reaction is transferred to a 96-well streptavidin-coatedplate containing 75 μL of deionized H₂O/well and is incubated at roomtemperature for 60 minutes. 11. The reaction mixture is washed threetimes with 200 μL/well PBS/T. 12. Dilution of primary antibody,phospho-IκB-α (Ser32/36) (5A5) Mouse mAb, 1:1000 in PBS/T with 1% BSA isprepared. A hundred μL/well primary antibody is added to the reactionmixture. 13. The mixture is incubated at room temperature for 120minutes. 14. The reaction mixture is washed three times with 200 μL/wellPBS/T. The reaction mixture is evaluated by colorimetric ELISA assay asfollows: 1. Appropriate dilution of horseradish peroxidase (HRP) labeledsecondary antibody in PBS/T with 1% BSA (1:500 dilution for anti-mouseIgG or 1:1000 for anti-rabbit IgG) is prepared. 2. A hundred μL/wellsecondary antibody solution is added to the reaction mixture which isobtained at 14. 3. The mixture is incubated at room temperature for 30minutes. 4. The mixture is washed five times with 200 μl/well PBS/T. 5.A hundred μL/well TMB (3,3′,5,5″-tetramethylbenzidine) substrate isadded to the resulting mixture. 6. The mixture is incubate at roomtemperature for 15 minutes. 7. A hundred μL/well of stop solution isadded, and then is mixed well. 8. The absorbance at 450 nm is measuredwith a microtiter plate reader.

Example 2 Synthesis and Characterization of MCE-1 and MCE-3

MCE-1 and MCE-3 were synthesized as described below and summarized inScheme 2.

Ethyl 1,4-dioxaspiro[4,5]decane-8-carboxilate (2). A mixture of ethyl4-oxocyclohexanecarboxylate (25 g), 10-campharsulfonic acid, ethyleneglycol (40 mL), and toluene (400 mL) was heated under reflux with aDean-Stark apparatus for 2.5 h (bath temp. 135° C.). The mixture wascooled down, and then was diluted with ether (200 mL). After theethylene glycol layer was separated, the organic layer was washed withsaturated aqueous sodium bicarbonate solution (2×) and brine (1×), driedover MgSO₄, and filtered. The filtrate was evaporated in vacuo to give 2as an oil (31.29 g, 99%): ¹H NMR (CDCl₃) δ 4.13 (2H, q, J=7.1 Hz), 3.95(4H, s), 2.33 (1H, m), 1.93 (2H, m), 1.79 (4H, m), 1.56 (2H, m) 1.25(3H, t, J=7.1 Hz); ¹³C NMR (CDCl₃) δ 175.4, 108.3, 64.5, 60.5, 41.8,33.9, 26.5, 14.4.

Ethyl 8-methyl-1,4-dioxa-spiro[4,5]decane-8-carboxylate (3). Ethyl1,4-dioxaspiro[4,5]decane-8-carboxilate (2) (5.8 g) was dissolved undera nitrogen atmosphere in dry THF (60 mL) and cooled in a dryice/isopropanol bath to −78° C. LDA (2M solution inTHF/n-heptane/ethylbenzene, 20 mL, 40 mmol) was added dropwise to thestirred solution and the mixture was allowed to warm to room temperaturefor 30 minutes. The reaction mixture was again cooled to −78° C. and toit was added a solution of iodomethane (5.5 g, 39 mmol) in dry THF (15mL) using a syringe pump over 20 minutes. This mixture was stirred at−78° C. for 1 h. The reaction mixture was allowed to reach roomtemperature. The reaction mixture was then diluted with ethyl ether (100mL) and the organic layer was washed with saturated aqueous ammoniumchloride solution (2×50 mL) and brine (1×50 mL), then dried overmagnesium chloride, filtered, and concentrated in vacuo to give ayellow-brown oil (8 g). The oil was purified by flash columnchromatography [petroleum ether-ethyl ether (2:1)] to give 3 (5.22 g,85%) as a colorless oil: ¹H NMR (CDCl₃) δ 4.14 (2H, q, J=7.1 Hz), 3.93(4H, s), 2.13 (2H, m), 1.64 (6H, m), 1.25 (3H, t, J=7.1 Hz), 1.18 (3H,s); ¹³C NMR (CDCl₃) δ 177.2, 108.7, 64.4, 60.6, 42.5, 33.1, 32.3, 26.2,14.4.

(8-Methyl-1,4-dioxa-spiro[4,5]dec-8-yl)-methanol (4). Under a nitrogenatmosphere, lithium aluminum hydride (31.2 mmol, 1.25 g, 2.43 eq.) wasadded to a stirred solution of 3 (12.84 mmol, 2.93 g) in anhydrous ethylether (275 mL) cooled to 0° C. in an ice bath. The resulting mixture wasstirred at room temperature for 1.5 hours. In order to quench remaininglithium aluminum hydride, water (2.48 mL) followed by 40% aqueous sodiumhydroxide solution (1.68 mL) and additional water (3.47 mL) weresuccessively added with stirring to the reaction mixture. The insolubleprecipitate was then removed by decanting and filtration. The filtratewas dried over magnesium sulfate, filtered, and concentrated in vacuo togive 4 (12.75 mmol, 2.28 g, 99%) as a yellow oil that was used in thenext reaction without further purification: ¹H NMR (CDCl₃) δ 3.94 (4H,s), 3.39 (2H, s), 1.68-1.35 (8H, m), 0.96 (3H, s); ¹³C NMR (CDCl₃) δ109.3, 71.8, 64.4, 34.5, 31.5, 30.7, 21.3.

8-Methyl-1,4-dioxa-spiro[4,5]decane-8-carbaldehyde (5). Under a nitrogenatmosphere, solid chromium (IV) oxide (84.7 mmol, 8.47 g, 6.6 eq.) wasadded to a stirred solution of extra dry pyridine (171 mmol, 14.53 mL,13.3 eq.) in dry methylene chloride (123 mL) cooled to 0° C. in an icebath. The resulting solution was stirred at room temperature for 15minutes. To this mixture was then added a solution of 4 (12.83 mmol,2.28 g) in dry methylene chloride (19 mL) and the resulting mixture wasstirred at room temperature for 30 minutes. The reaction mixture wasthen decanted into a seperatory funnel and the residue was washed withethyl ether (40 mL and 50 mL). The washings were combined and theorganic layer was washed with 5% aqueous sodium hydroxide solution (2×40mL), 5% aqueous hydrochloric acid solution (3×40 mL), saturated aqueoussodium bicarbonate solution (3×40 mL) and brine (3×40 mL), then driedover magnesium sulfate, filtered, and concentrated in vacuo to give 5(12.24 mmol, 2.35 g, 95%) as an amorphous solid. The product was used inthe next reaction without further purification: ¹H NMR (CDCl₃) δ 9.47(1H, s), 3.95 (4H, s), 2.00-1.96 (2H, m), 1.71-1.67 (2H, m), 1.61-1.53(4H, m), 1.05 (s, 3H); ¹³C NMR (CDCl₃) δ 205.9, 108.4, 64.4, 64.4, 45.7,31.4, 29.9, 21.2.

Trimethyl((8-methyl-1,4-dioxaspiro[4.5]decan-8-yl)ethynyl)silane (6) Toa suspension of chloromethylene triphenylphosphonyl chloride (95%, 24.2g, 66 mmol) in THF (72 mL) was added n-BuLi (1.6 M in hexane, 41.5 mL,66 mmol) dropwise in an ice-water bath. To the mixture was added HMPA(11.8 mL). The mixture was stirred at room temperature for 20 min. Tothe mixture was added a solution of 5 (3.06 g, 16.6 mmol) in THF (72mL). The mixture was stirred at room temperature for 1 h. To the mixturewas added saturated aqueous ammonium chloride solution (600 mL) Theaqueous mixture was extracted with methylene chloride/ether (1:2, 300mL×3). The extract was washed with brine (×2), dried over MgSO₄, andfiltered. The filtrate was evaporated in vacuo to give a browncrystalline solid (21.31 g). The solid was washed with ether severaltimes. The ether solution was evaporated in vacuo to give a crystallinesolid (9.78 g). The solid was purified by flash chromatography[petroleum ether-ether (5:1)] to give8-(2-chlorovinyl)-8-methyl-1,4-dioxaspiro[4.5]decane (2.74 g, 76%). To asolution of the solid in THF (340 mL) was added MeLi (1.6 M in ether,100 mL, 160 mmol) dropwise in an ice-water bath. The mixture was stirredat room temperature over night. To the reaction mixture was added water(500 mL). The aqueous mixture was extracted with methylenechloride/ether (1:2, 300 mL×3). The extract was washed with saturatedaqueous sodium bicarbonate solution (300 mL×1) and brine (300 mL×2),dried over MgSO4, and filtered. The filtrate was evaporated in vacuo togive an oil (3.2 g). The oil was purified by flash chromatography togive 6 as a crystalline solid (2.86 g, 90%): ¹H NMR (CDCl₃) δ 3.94 (4H,m), 1.95, 1.73, 1.61, 1.50 (each 2H, m), 1.22 (3H, s), 0.14 (9H, s); ¹³CNMR (CDCl₃) δ 112.8, 108.9, 85.1, 64.4, 36.9, 32.7, 32.2, 29.4, 0.5.

4-Methyl-4-((trimethylsilyl)ethynyl)cyclohexanone (7). To a solution of6 (2.1 g, 8.3 mmol) in acetone (25 mL) was added 10% aqueous HClsolution (15 mL). The solution was cloudy. Thus, to the cloudy solution,was acetone dropwise (total 12 mL) until the solution was clear. Theclear solution was stirred at room temperature for 5 h. The mixture wasdiluted with brine (250 mL). The aqueous mixture was extracted withmethylene chloride/ether (1:2, 200 mL×3). The extract was washed withsaturated aqueous bicarbonate solution (2×100 mL) and brine (100 mL×1),dried over MgSO₄, and filtered. The filtrate was evaporated in vacuo togive 7 as a crystalline solid (1.7 g, 98%): ¹H NMR (CDCl₃) δ 2.75, 2.29,2.04, 1.65 (each 2H, m), 1.32 (3H, s), 0.17 (9H, s); ¹³C NMR (CDCl₃) δ211.8, 110.8, 86.9, 39.2, 38.8, 32.9, 28.8, 0.4.

4-Methyl-4-((trimethylsilyl)ethynyl)cyclohex-2-enone (8). To a solutionof 7 (260 mg, 1.25 mmol) in THF (10 mL) was added LDA (2 M inTHF/n-heptane/ethylbenzene, 935 μL, 1.87 mmol) at −78° C. (in anisopropanol-dry ice bath). The mixture was allowed to reach roomtemperature over 20 min. To the mixture was added a solution ofphenylselenyl chloride (472 mg, 2.5 mmol) in THF (3 mL) at −78° C. Themixture was stirred at room temperature for 2 h. The reaction mixturewas quenched with brine (10 mL). The aqueous mixture was extracted withmethylene chloride/ether (1:2, 50 mL×3). The extract was washed withbrine (50 mL×1), dried over MgSO₄, and filtered. The filtrate wasevaporated in vacuo to give a brown residue (758 mg). To a solution ofthe residue in methylene chloride (20 mL) was added 30% aqueous hydrogenperoxide (0.3 mL). Five minutes after the addition, 30% aqueous hydrogenperoxide (0.3 mL) was added again. The brown color became to a paleyellow. Five minutes later, the reaction mixture was washed with water(5 mL×1), saturated aqueous sodium bicarbonate solution (5 mL×2), andbrine (5 mL×1), dried over MgSO₄, and filtered. The filtrate wasevaporated in vacuo to give a residue (290 mg). The residue was purifiedby flash chromatography [petroleum ether-ether (5:1)] to give 8 as acrystalline solid (91.5 mg, 36%): ¹H NMR (CDCl₃) δ 6.69 (1H, dd, J=1.5and 10 Hz), 5.88 (1H, d, J=10 Hz), 2.73 (1H, m), 2.44 (1H, m), 2.22 (1H,m), 1.95 (1H, m), 1.43 (3H, s), 0.14 (9H, s); ¹³C NMR (CDCl₃) δ 199.2,153.7, 127.6, 107.8, 99.4, 36.7, 35.2, 28.2, 0.2.

5-Methyl-5-((trimethylsilyl)ethynyl)-4,5-dihydrobenzo[d]isoxazole (9).To a solution of 8 (139 mg, 0.67 mmol) in dry benzene (7.8 mL) wereadded ethyl formate (97%, 244 mg, 3.2 mmol) and sodium methoxide (176mg, 3.3 mmol), successively. The mixture was stirred at room temperaturefor 1 h. The mixture was diluted with methylene chloride/ether (1:2, 40mL). The mixture was washed with 5% aqueous HCl solution (15 mL×2). Theacidic washings were extracted with methylene chloride/ether (1:2, 20mL×1). The original organic solution and the extract were combined. Thecombined solution was washed with water (15 mL×2) and brine (15 mL×1),dried over MgSO₄, and filtered. The filtrate was evaporate in vacuo togive6-(hydroxymethylene)-4-methyl-4-((trimethylsilyl)-ethynyl)cyclohex-2-enone(148 mg, 94%): ¹H NMR (CDCl₃) δ 13.69 (1H, brs), 7.53 (1H, s), 6.64 (1H,d, J=9.9 Hz), 6.00 (1H, d, J=9.9 Hz), 2.78 (1H, d, J=14.6 Hz), 2.47 (1H,d, J=14.6 Hz), 1.38 (3H, s), 0.14 (9H, s); ¹³C NMR (CDCl₃) δ 187.7,168.0, 152.3, 126.8, 110.2, 109.5, 106.1, 37.2, 37.1, 26.9, 0.2. Thismaterial was used without further purification for the next step.

To a solution of6-(hydroxymethylene)-4-methyl-4-((trimethylsilyl)ethynyl)-cyclohex-2-enone(147 mg, 0.63 mmol) in ethanol (15 mL) was added a solution ofhydroxylamine hydrochloride (452 mg) in water (0.65 mL). The mixture washeated under reflux for 1 h. To the mixture was added water (50 mL). Theaqueous mixture was extracted with methylene chloride/ether (1:2, 50mL×3). The extract was washed with saturated aqueous sodium bicarbonatesolution (50 mL×1) and brine (50 mL×1), dried over MgSO₄, and filtered.The filtrate was evaporated in vacuo to give 9 as a crystalline solid(145 mg, quantitative yield): ¹H NMR (CDCl₃) δ 8.06 (1H, s), 6.47 (1H,d, J=9.9 Hz), 6.03 (1H, d, J=9.9 Hz), 3.07 (1H, d, J=16.1 Hz), 2.77 (1H,d, J=16.1 Hz), 1.37 (3H, s), 0.14 (9H, s). This material was usedwithout further purification for the next step.

5-Ethynyl-5-methyl-2-oxocyclohex-3-enecarbonitrile (MCE-4, dhMCE-1). Toa solution of 9 (75 mg, 0.32 mmol) in dry ether (7.4 mL) was added asolution of sodium methoxide (565 mg) in dry methanol (6 mL). Themixture was stirred at room temperature for 1 h. The mixture was dilutedwith methylene chloride/ether (1:2, 40 mL). The mixture was washed with5% aqueous HCl solution (10 mL×2). The acidic washings were extractedwith methylene chloride/ether (1:2, 10 mL×1). The original organicsolution and the extract were combined. The combined solution was washedwith water (15 mL×2) and brine (15 mL×1), dried over MgSO₄, andfiltered. The filtrate was evaporated in vacuo to give MCE-4 as acrystalline solid (51 mg, quantitative yield). This material was usedwithout further purification for the next step.

3-Ethynyl-3-methyl-6-oxocyclohexa-1,4-dienecarbonitrile (MCE-1). Asolution of MCE-4 (47.7 mg, 0.3 mmol) and DDQ (102 mg, 0.45 mmol), indry benzene (3 mL) was heated under reflux for 15 min. After removal ofinsoluble matter, the filtrate was concentrated in vacuo to give aresidue (149 mg). The residue was purified by flash chromatography[hexanes-ethyl acetate (2:1)] and subsequent preparative TLC[hexanes-ethyl acetate (2:1)] to give MCE-1 as a crystalline solid (22.3mg, 47%): ¹H NMR (CDCl₃) δ 7.54 (1H, d, J=2.9 Hz), 6.96 (1H, dd, J=2.9and 9.9 Hz), 6.34 (1H, d, J=9.9 Hz), 2.44 (1H, s), 1.65 (3H, s); ¹³C NMR(CDCl₃) δ 177.9, 160.0, 150.6, 126.6, 116.5, 113.5, 79.2, 73.5, 36.0,27.2.

3-Methyl-6-oxo-3-((trimethylsilyl)ethynyl)cyclohex-1-enecarbonitrile(10). To a solution of 7 (163 mg, 0.78 mmol) in THF (8.8 mL) was addedLDA (2M solution in THF/n-heptane/ethylbenzene, 1.08 mL, 2.16 mmol) at−78° C. (in an isopropanol-dry ice bath). The mixture was allowed toreach room temperature over 20 min. To the mixture was added a cloudysolution of p-toluenesulfonyl cyanide (95%, 591 mg, 3.1 mmol) in THF(6.8 mL) at −78° C. The mixture was stirred at −78° C. for 30 min. Tothe reaction mixture was added saturated aqueous ammonia solution (4.5mL) at −78° C. The mixture was allowed to reach room temperature. Themixture was acidified with 10% aqueous HCl solution. The mixture wasextracted with ethyl acetate (30 mL×3). The extract was washed withsaturated aqueous sodium bicarbonate solution (40 mL×2) and brine (40mL), dried over MgSO₄, and filtered. The filtrate was evaporated invacuo to give5-methyl-2-oxo-5-((trimethylsilyl)ethynyl)cyclohexanecarbonitrile (270mg).

A solution of5-methyl-2-oxo-5-((trimethylsilyl)ethynyl)cyclohexane-carbonitrile (270mg) and DDQ (350 mg) in benzene (20 mL) was heated under reflux for 10min. After removal of insoluble matter, the filtrate was concentrated invacuo to give a residue (407 mg). The residue was purified by flashchromatography [hexanes-ethyl acetate (4:1)] to give 10 as a crystallinesolid (142 mg, 78%): ¹H NMR (acetone-d₆) δ 7.71 (1H, d, J=1.5 Hz), 2.72(1H, m), 2.62 (1H, m), 2.21 (2H, m), 1.55 (3H, s), 0.15 (9H, s); ¹³C NMR(acetone-d₆) δ 192.0, 164.8, 116.4, 114.8, 106.5, 88.7, 35.9, 35.0, 0.0.

3-Ethynyl-3-methyl-6-oxocyclohex-1-enecarbonitrile (MCE-3). A solutionof 10 (20 mg, 0.086 mmol) in THF (0.6 mL) was degassed by gentle argonstream for 5 min. To the solution was added tetra-(n-butyl)ammoniumfluoride (TBAF, 70 mg). The mixture was stirred at room temperature for20 min. The mixture was diluted with methylene chloride/ether (1:2, 20mL). The mixture was extracted with saturated aqueous sodium bicarbonatesolution (10 mL×3). The basic extract was acidified with 5% aqueous HClsolution. The acidic mixture was extracted with methylene chloride/ether(1:2, 10 mL×4). The extract was washed with water (10 mL×2), dried overMgSO₄, and filtered. The filtrate was evaporated in vacuo to give acrystalline solid. The solid was purified by preparative TLC[hexanes-ethyl acetate (2.5:1)] to give MCE-3 as a crystalline solid(6.9 mg, 50%): ¹H NMR (CDCl₃) δ 7.39 (1H, d, J=1.8 Hz), 2.84 (1H, m),2.62 (1H, m), 2.38 (1H, s), 2.32 (1H, m), 2.05 (1H, m), 1.55 (3H, s).

Example 3 Synthesis and Characterization of MCE-5

MCE-5 was synthesized as described below and summarized in Scheme 3.

2,2,4,4-Tetramethylcyclohexanone (2). A three-necked flask fitted withan additional funnel and dry ice condenser was dried in vacuo withheating. Under a nitrogen atmosphere, liquid ammonia (45 mL) wascollected in the flask from condensation of ammonia gas. The flask wascooled to −78° C. in an isopropanol/dry ice bath and to it was addedfreshly cut lithium wire (108 mg, 15.5 mmol, 2.6 eq) until a blue colorpersisted. The mixture was stirred at −78° C. for 15 minutes. A solutionof 14 (829.3 mg, 6 mmol) and tert-butanol (445 mg, 6 mmol, 1 eq) inanhydrous ethyl ether (24 mL) was then added dropwise over fifteenminutes. When the solution was completely added, the isopropanol bathwas removed and the reaction mixture was allowed to warm to roomtemperature and begin to reflux. The solution was then cooled to −33° C.in a carbon tetrachloride (CCl₄)/dry ice bath and the reaction wasstirred at reflux for 45 minutes, after which the CCl₄ bath was removed.At this time, the blue color still persisted, indicating an excess oflithium. Therefore, isoprene (0.2 mL) was added to the reaction mixtureuntil the blue color disappeared. The reaction mixture was then cooledto −78° C. and a solution of methyl iodide (2.24 mL, 36 mmol, 6 eq) inanhydrous ethyl ether (15 mL) was added dropwise over 15 minutes. Theisopropanol bath was again removed and when the reaction mixture beganto reflux, it was placed in a CCl₄ bath and stirred at reflux for 1hour. The CCl₄ bath was then removed. Finally, the liquid ammonia wasremoved with the aid of a nitrogen stream and remaining ammonia wasneutralized by addition of 10% aqueous hydrochloric acid solution (25mL). The resulting solution was extracted with methylene chloride/ethylether (1:2, 5×30 mL), and the organic phases were combined and washedwith saturated aqueous sodium bicarbonate solution (1×40 mL) and brine(1×40 mL), dried over magnesium sulfate, filtered, and concentrated invacuo to give crude 15 (724.1 mg) as an orange-brown liquid. The crudeproduct was purified by flash column chromatography [hexanes-ethylacetate (9:1)] to give 15 (461 mg, 2.98 mmol, 50%) as a yellow-brownoil: ¹H NMR (CDCl₃) δ 2.44 (2H, t, J=6.8 Hz), 1.70 (2H, t, J=6.8 Hz),1.59 (2H, s), 1.12 (6H, s), 1.08 (6H, s); ¹³C NMR (CDCl₃) δ 217.7, 53.5,38.8, 35.4, 30.8, 30.3, 28.0.

6-Hydroxymethylene-2,2,4,4-tetramethylcyclohexanone (16). Under an argonatmosphere, ethyl formate (1.18 mL, 14.3 mmol, 11 eq) then sodiummethoxide (772.5 mg, 14.3 mmol, 11 eq) were successively added to asolution of 15 (200 mg, 1.30 mmol) in dry benzene (5.83 mL). Thismixture was stirred at room temperature for 1 hour, during which time acolor change to a cloudy, pale yellow solution occurred. The mixture wasthen diluted with a mixture of methylene chloride/ethyl ether (1:2, 20mL) and washed with saturated aqueous ammonium chloride solution (2×8mL). The aqueous washings were combined and extracted with methylenechloride/ethyl ether (1:2, 3×8 mL). The organic layers were thencombined and washed with brine (1×8 mL), dried over magnesium sulfate,filtered, and concentrated in vacuo to give 16 as a yellow liquid (207.6mg, 1.14 mmol, 88%). The product was used in the next reaction withoutfurther purification: ¹H NMR (CDCl₃) δ 14.82 (1H, d, J=4 Hz), 8.42 (1H,d, J=5 Hz), 2.12 (2H, s), 1.45 (2H, s), 1.22 (6H, s), 0.99 (6H, s); ¹³CNMR (CDCl₃) δ 193.6, 185.1, 106.8, 50.8, 38.4, 38.0, 30.6, 29.3, 29.2.MS: No molecular ion peak is observed by ESI+ and EI methods.

5,5,7,7-Tetramethyl-4,5,6,7-tetrahydrobenzo[d]isoxazole (17). Under anitrogen atmosphere and with stirring, a solution of 16 (140 mg, 0.77mmol) in ethanol (19.1 mL) was added to a solution of hydroxylaminehydrochloride (7.98 mmol, 555 mg, 10.4 eq) in water (980 μL) at roomtemperature. The mixture was heated to 110° C. and stirred at reflux for1 hour. After heating, the reaction mixture was diluted with water (10mL) and the aqueous layer was extracted with methylene chloride/ethylether (1:2, 3×10 mL). The organic layers were then combined and washedwith saturated aqueous sodium bicarbonate solution (1×12 mL) and brine(2×12 mL), dried over magnesium sulfate, filtered, and concentrated invacuo to give 17 as a yellow oil (122 mg, 0.68 mmol, 89%). The materialwas used in the next reaction without further purification: ¹H NMR(CDCl₃) δ 8.00 (1H, s), 2.24 (2H, s), 1.60 (2H, s), 1.34 (6H, s), 1.03(6H, s); ¹³C NMR (CDCl₃) δ 173.2, 150.0, 110.4, 52.4, 34.4, 32.6, 32.4,29.7, 29.3. MS: No molecular ion peak is observed by ESI+ and EImethods.

3,3,5,5-Tetramethyl-2-oxocyclohex-1-enecarbonitrile (MCE-5). Under anargon atmosphere and with stirring, a solution of 17 (152 mg, 0.848mmol) in anhydrous ethyl ether (19.5 mL) was added to a solution ofsodium methoxide (687 mg, 12.7 mmol, 15 eq) in dry methanol (15.8 mL).The mixture was stirred at room temperature for 2 hours, giving a lightyellow solution. The reaction mixture was then diluted with methylenechloride/ethyl ether (1:2, 30 mL) and washed with 5% aqueoushydrochloric acid solution (2×7 mL). The aqueous layers were combinedand extracted with methylene chloride/ethyl ether (1:2, 3×15 mL). Theorganic layers were then combined and washed with water (2×10 mL) andbrine (1×10 mL), dried over magnesium sulfate, filtered, andconcentrated in vacuo to give dhMCE-5 as a yellow oil (136.1 mg, 90%).

The calculations for the next step of the reaction were performedassuming the previous step gave a quantitative yield.2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) (148.6 mg, 0.642 mmol, 1 eq)was introduced into a flask containing dhMCE-5 (115 mg, 0.642 mmol), andthe contents of the flask were dissolved under a nitrogen atmosphere indry benzene (18.6 mL). The mixture was heated under reflux for 20minutes. The reaction mixture was then allowed to cool to roomtemperature and was filtered through a Pasteur pipette with cotton andrinsed with benzene. The resulting solution was concentrated in vacuo togive a dark red solid as the crude product. The crude was purified byflash column chromatography [hexanes:ethyl acetate (4:1)] to give MCE-5(35.0 mg, 0.197 mmol, 31%) as a white solid: ¹H NMR (CDCl₃) δ 7.32 (1H,s), 1.83 (2H, d, J=1 Hz), 1.27 (6H, s), 1.20 (6H, s); ¹³C NMR (CDCl₃) δ197.5, 169.0, 114.8, 113.7, 48.2, 41.5, 34.4, 29.9, 26.9. MS (ESI+) m/z178.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₁H₁₅NO+H 178.1232. found 178.1227.

Example 4 Synthesis and Characterization of MCE-15

MCE-15 was synthesized as described below and summarized in Scheme 4.

8-Methyl-8-vinyl-1,4-dioxaspiro[4,5]decane (18). In a three-necked flaskunder a nitrogen atmosphere, potassium tert-butoxide (73.2 mmol, 8.21 g,7.93 eq.) was added to a stirred solution of methyltriphenylphosphoniumiodide (87.3 mmol, 35.7 g, 9.73 eq.) in dry THF (130 mL), and theresulting mixture was stirred at room temperature for 5 minutes. To themixture was added a solution of 5 (9.23 mmol, 1.7 g) in dry THF (130 mL)and the resulting mixture was stirred at room temperature for 1 hour.The mixture was then diluted with water (255 mL) and extracted withmethylene chloride/ethyl ether (1:2, 3×300 mL). The organic layers werecombined and washed with brine (2×350 mL), then dried with magnesiumsulfate, filtered, and concentrated in vacuo to give a yellow solid(13.4 g). The crude product was purified by washing with petroleumether-ethyl ether (2:1, 5×75 mL washes) and separating the solublecompound from the insoluble reaction residue. The washes were thencombined, filtered, and concentrated in vacuo, revealing residualinsoluble material in the form of white crystals. The soluble materialwas therefore decanted, and the crystals were washed with petroleumether-ethyl ether (2:1, 2×50 mL washes). The washes were again combined,filtered, and concentrated in vacuo to give 18 (8.5 mmol, 1.55 g, 92%)as a yellow residue: ¹H NMR (CDCl₃) δ 5.79 (1H, dd, J=13.5, 18 Hz), 5.03(1H, dd, J=1.2, 4.5 Hz), 4.98 (1H, dd, J=1.2, 2.7 Hz), 3.94 (4H, s),1.70-1.61 (6H, m), 1.55-1.46 (2H, m), 1.01 (3H, s); ¹³C NMR (CDCl₃) δ147.2, 111.6, 109.3, 64.4, 64.3, 35.9, 34.8, 31.4. MS: No molecular ionpeak is observed by ESI+ and EI methods.

4-Methyl-4-vinylcyclohexanone (19). To a stirred solution of 2 (1.98mmol, 360 mg) in acetone (7.76 mL) was added 10% aqueous hydrochloricacid solution (3.02 mL), and the resulting mixture was stirred at roomtemperature for 2.5 hours. The reaction mixture was then diluted withbrine (40 mL) and extracted with methylene chloride/ethyl ether (1:2,3×30 mL). The organic layers were combined and washed with saturatedaqueous sodium bicarbonate solution (2×30 mL) and brine (1×30 mL), thendried over magnesium sulfate, filtered, and concentrated in vacuo togive 19 (251.6 mg, 1.92 mmol, 94%) as a yellow oil that was used in thenext reaction without further purification: ¹H NMR (CDCl₃) δ 5.89 (1H,dd, J=0.5, 17.3 Hz), 5.15 (1H, dd, J=1, 3.5 Hz), 5.12 (1H, dd, J=0.5, 10Hz), 2.42-2.28 (4H, m), 1.97-1.91 (2H, m), 1.74-1.68 (2H, m), 1.12 (3H,s); ¹³C NMR (CDCl₃) δ 212.4, 145.5, 112.9, 38.2, 37.1, 27.4; MS (EI) m/z138.1 [M⁺], 123.1, 110.1, 105.1, 97.1, 86.0 (100%), 68.1, 63.0, 54.9;HRMS (EI) calcd for C₉H₁₄O 138.1045 found 138.1048.

4-Methyl-4-vinylcyclohex-2-enone (20). Under a nitrogen atmosphere, astirred solution of 19 (123.8 mg, 0.899 mmol) in dry THF (7.12 mL) wascooled to −78° C. in a dry ice/isopropanol bath and to it was added LDA(2M in THF/n-heptane/ethylbenzene, 672 μL, 1.34 mmol, 1.5 eq.). The dryice bath was then removed and the mixture was allowed to warm to roomtemperature for 20 minutes. To the mixture was added a solution ofphenylselenenyl chloride (350 mg, 1.79 mmol, 2 eq.) in dry THF (2.2 mL)and the resulting mixture was stirred at room temperature for 2 hours.The reaction was quenched with brine (10 mL) and then extracted withmethylene chloride/ethyl ether (1:2, 3×15 mL). The organic layers werecombined and washed with brine (1×25 mL), then dried over magnesiumsulfate, filtered, and concentrated in vacuo to give a yellow oil. Thisresidue was then dissolved in methylene chloride (13.5 mL) and to it wasadded 3 successive portions of hydrogen peroxide (3×210 μL), allowingthe mixture to stir 5 minutes at room temperature between each addition.After the third portion, the solution became light yellow and opticallyclear. The mixture was then diluted with ethyl ether (20 mL) and theorganic layer was washed with water (1×10 mL), saturated aqueous sodiumbicarbonate solution (2×10 mL) and brine (1×10 mL), then dried overmagnesium sulfate, filtered, and concentrated in vacuo to give a yellowoil (153.8 mg). The crude product was combined with the crude from aprevious identical reaction (131.7 mg) and purified by flash columnchromatography [hexanes-ethyl acetate (7:1)] to give a yellow oil (62.1mg). When the oil was analyzed by ¹H NMR, starting material 19 was foundto exist with the desired product 20 in a 1:1.4 ratio. Therefore, thepurified product included 0.204 mmol of 20, giving 16% yield overall. ¹HNMR (CDCl₃) δ 6.63 (1H, d, J=10 Hz), 5.99 (1H, d, J=10.2 Hz), 5.83 (1H,dd, J=6.5, 30 Hz), 5.12 (1H, ddd, J=1, 13, 18 Hz), 5.02 (1H, dd, J=1, 30Hz), 2.5-2.26 (4H, m), 1.25 (3H, s); ¹³C NMR (CDCl₃) δ 156.1, 142.7,128.7, 114.5, 38.3, 34.9, 34.4, 27.2.

6-Hydroxymethylene-4-methyl-4-vinyl-cyclohex-2-enone (21) and6-hydroxymethylene-4-methyl-4-vinyl-cyclohexanone (22). The product ofthe previous reaction (62.1 mg) was combined with the purified productof a subsequent identical reaction and purification sequence (455 mg)that was also found to contain 19 (starting material) in a 1:1.4 ratiowith 20. To a solution of this starting material (517 mg, 1.56 mmol 3,2.18 mmol 20) under an argon atmosphere in dry benzene (39 mL) was addedethyl formate (1.9 g, 26 mmol, 10 eq. per mol 20, 5 eq. per mol 19(double)) followed by sodium methoxide (1.3 g, 24 mmol, 10 eq. per mol20, 5 eq. per mol 19), and the resulting solution was stirred at roomtemperature for 1 hour. The reaction mixture was then transferred to aseperatory funnel, and to it was added 5% aqueous hydrochloric acidsolution and methylene chloride/ethyl ether (1:2) until a separation oflayers was observed. The organic layer was then washed with 5% aqueoushydrochloric acid solution, and the combined aqueous washings wereextracted with methylene chloride/ethyl ether (1:2, 3×). The organiclayers were then combined and washed with brine (3×), dried overmagnesium sulfate, filtered, and concentrated in vacuo to give an oil(500 mg). The crude product was purified by flash column chromatography[petroleum ether-ethyl ether (5:1) followed by petroleum ether-ethylether (3:1) to elute remaining product] to obtain 22 (114 mg, 0.686mmol, 43% yield from 19) and 5 (183.6 mg, 1.118 mmol, 50% yield from 20)as separate fractions.

21: ¹H NMR (CDCl₃) δ 13.72 (1H, bs), 7.45 (1H, bs), 6.48 (1H, d, J=10Hz), 6.06 (1H, d, J=10 Hz), 5.80 (1H, dd, J=10.6, 17.2 Hz), 5.06 (1H,dd, J=1, 4.4 Hz), 5.01 (J=1, 11 Hz), 2.42 (2H, s), 1.22 (3H, s); ¹³C NMR(CDCl₃) δ 188.8, 167.2, 164.6, 154.4, 143.0, 127.6, 113.7, 39.7, 36.2,25.7; MS (ESI+) m/z 165.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₀H₁₂O₂+H165.0916. found 165.0908.

22: ¹H NMR (CDCl₃) δ 14.41 (1H, bs), 8.60 (1H, s), 5.78 (1H, dd, J=10.6Hz, 17.2 Hz), 5.03 (1H, dd, J=1, 4 Hz), 4.98 (1H, dd, J=1.1, 10.6 Hz),2.39-2.16 (4H, m), 1.70-1.50 (2H, m), 1.07 (3H, s); ¹³C NMR (CDCl₃) δ187.3, 184.8, 145.4, 112.4, 107.6, 35.6, 34.5, 32.2, 28.9, 26.2. MS: Nomolecular ion peak is observed by ESI+ and EI methods.

5-methyl-5-vinyl-4,5-dihydro-benzo[d]isoxazole (23). To a stirredsolution of 21 (177 mg, 1.078 mmol) in ethanol (15 mL) under a nitrogenatmosphere was added a solution of hydroxylamine hydrochloride (741 mg,10.66 mmol, 10 eq.) in water (1 mL). The reaction mixture was heated toreflux at 115° C. for 1 hour. Water was then added to the mixture untilit became cloudy and the aqueous mixture was extracted with methylenechloride/ethyl ether (1:2, 4×). The organic layers were then combinedand washed with saturated sodium bicarbonate solution (1×) and brine(1×), dried over magnesium sulfate, filtered and concentrated in vacuoto give 23 (159.7 mg, 0.99 mmol, 92%) as an oil. The product was used inthe next reaction without further purification: ¹H NMR (CDCl₃) δ 8.03(1H, s), 6.52 (1H, dd, J=0.6, 10 Hz), 5.87 (1H, d, J=10 Hz), 5.81 (1H,d, J=10.6 Hz), 5.04 (1H, dd, J=0.6, 18.3 Hz), 5.00 (1H, dd, J=1.2, 11.4Hz), 2.69 (2H, dd, J=16.2, 34.8 Hz) 1.23 (3H, s); ¹³C NMR (CDCl₃) δ165.0, 148.8, 143.6, 141.6, 114.4, 112.7, 109.5, 40.4, 31.4, 26.0; MS(ESI+) m/z 162.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₀H₁₁NO+H 162.0919.found 169.0913.

3-methyl-6-oxo-3-vinyl-cyclohexa-1,4-dienecarbonitrile (MCE-15). Underan argon atmosphere, a solution of 23 (155 mg, 0.962 mmol) in anhydrousethyl ether (17 mL) was added to a stirred solution of sodium methoxide(1.6 g, 29.61 mmol, 30 eq.) in dry methanol and the resulting mixturewas stirred at room temperature for 1 hour. The reaction mixture wasthen diluted with methylene chloride/ethyl ether (1:2, 120 mL) and thesolution was washed with 5% aqueous hydrochloric acid solution (2×). Theacidic washings were combined and extracted with methylenechloride/ethyl ether (1:2, 30 mL). All of the organic layers were thencombined and washed with water (2×) and brine (1×), dried over magnesiumsulfate, filtered, and concentrated in vacuo to give 24 as a yellow oil(148.8 mg, 96%).

Calculations for the next step of the reaction were done assuming thatthe previous step gave a quantitative yield. Under a nitrogenatmosphere, the yellow oil (80 mg, 0.5 mmol) was dissolved in drybenzene (4 mL) and to it was added DDQ (150 mg, 0.661 mmol, 1.33 eq).The reaction mixture was heated to reflux for 10 minutes. When checkedby thin layer chromatography, some starting material still remained.Therefore, additional DDQ (40 mg, 0.176 mmol) was added and the reactionmixture was again stirred at reflux for 10 minutes. The reaction wasallowed to cool to room temperature, and insoluble material was removedby filtration through a Pasteur pipette with cotton and washing withbenzene. The filtrate was concentrated in vacuo to give an orangeresidue (400 mg). The crude residue was then purified by flash columnchromatography [petroleum ether-ethyl ether (1:1)] to give MCE-15 (48mg, 0.301 mmol, 61%) as a crystalline solid: ¹H NMR (CDCl₃) δ 7.51 (1H,d, J=3 Hz), 6.90 (1H, dd, J=3, 10.2 Hz), 6.34 (1H, d, J=10.2 Hz), 5.73(1H, dd, J=10.2, 17.7 Hz), 5.27 (1H, d, J=10.5 Hz), 5.21 (1H, d, J=17.4Hz), 1.46 (3H, s); ¹³C NMR (CDCl₃) δ 179.0, 163.8, 153.8, 136.7, 126.8,110.1, 116.1, 114.0, 45.0, 24.1; MS (ESI+) m/z 160.1 [M+H]⁺, HRMS (ESI+)calcd for C₁₀H₉NO+H 160.0762. found 160.0755.

Example 5 Synthesis and Characterization of MCE-13

MCE-13 was synthesized as described below and summarized in Scheme 5.

Ethyl 8-benzyl-1,4-dioxa-spiro[4,5]decane-8-carboxylate (25). Under anitrogen atmosphere, LDA (2M in THF/n-heptane/ethylbenzene, 20 mL, 40mmol, 1.5 eq.) was added dropwise with stirring to a solution of 2 (5.8g, 27 mmol) in dry tetrahydrofuran (THF, 60 mL) cooled to −78° C. in adry ice/isopropanol bath. The mixture was allowed to slowly warm to roomtemperature over 20 minutes. The reaction mixture was again cooled to−78° C. and to it was added a solution of benzyl bromide (5.42 g, 32mmol, 1.17 eq.) in dry THF (15 mL) using a syringe pump over 20 minutes.The reaction mixture was then stirred at −78° C. in the dryice/isopropanol bath for 1 hour. After warming to room temperature, themixture was diluted with ethyl ether (100 mL) and the organic layer waswashed with saturated aqueous ammonium chloride solution (2×50 mL) andbrine (1×50 mL) then dried over magnesium sulfate, filtered, andconcentrated in vacuo to give a brown residue (14.9 g) as a crude. Thecrude product was purified by flash column chromatography [hexanes-ethylacetate (4:1)] to give 25 (6.67 g, 81%) as an oil. ¹H NMR (CDCl₃) δ7.28-7.18 (3H, m), 7.07-7.04 (2H, m), 4.08 (4H, q, J=7.1 Hz), 3.94 (4H,s), 2.83 (2H, s), 2.17-2.14 (2H, m), 1.72-1.53 (6H, m); ¹³C NMR (CDCl₃)δ 175.5, 137.3, 130.0, 128.2, 126.8, 108.8, 64.5, 64.4, 60.5, 47.9,46.7, 33.9, 32.3, 31.7, 26.5, 14.3; MS (ESI+) m/z 305.2 [M+H]⁺, HRMS(ESI+) calcd for C₁₈H₂₄O₄+H 305.1753. found 305.1753.

(8-Benzyl-1,4-dioxa-spiro[4,5]dec-8-yl)-methanol (26). A sample of 25(687.6 mg, 2.26 mmol) was dissolved in anhydrous ethyl ether (48.8 mL)at 0° C. under a nitrogen atmosphere and with stirring. To this wasadded lithium aluminum hydride (214.4 mg, 5.64 mmol, 2.5 eq.), and theresulting mixture was stirred at room temperature for 2 hours. Afterverifying the completion of the reaction by thin layer chromatography,water (3.6 mL), 40% aqueous sodium hydroxide solution (2.56 mL), andadditional water (5.2 mL) were added successively to the mixture whilestirring to quench excess lithium aluminum hydride. The resultingsolution was filtered, then dried over magnesium sulfate, filteredagain, and concentrated in vacuo to give a colorless oil (1.50 g). Thecrude product was combined with the crude from a previous identicalreaction (84.8 mg) and purified by flash column chromatography[hexanes-ethyl acetate (5:1)] to give 26 (609.6 mg, 2.32 mmol, 91%) asan oil: ¹H NMR (CDCl₃) δ 7.31-7.19 (5H, m), 3.96 (4H, t, J=2.3 Hz), 3.37(2H, d, J=4.8 Hz), 2.71 (2H, s), 1.81-1.51 (8H, m), 1.34 (1H, bs); ¹³CNMR (CDCl₃) δ138.6, 130.6, 128.3, 126.3, 109.2, 66.3, 64.5, 40.7, 30.7,30.0; MS (ESI+) m/z 263.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₆H₂₂O₃+H263.1647. found 263.1640.

8-benzyl-1,4-dioxa-spiro[4,5]decane-8-carbaldehyde (27). Under anitrogen atmosphere and with stirring solid chromium (IV) oxide (140 mg,1.40 mmol, 7 eq.) was added to a solution of extra dry pyridine (220 μL,2.72 mmol, 13.6 eq.) in dry methylene chloride (2.44 mL) cooled to 0° C.in an ice bath. The resulting mixture was stirred at room temperaturefor 15 minutes, and to it was added a solution of 3 (52.0 mg, 0.2 mmol)in dry methylene chloride (0.4 mL+(3×5 mL) rinse). After stirring atroom temperature for 30 minutes, the reaction mixture was decanted intoa seperatory funnel and the residue was washed with ethyl ether (1.67mL+1.3 mL+1.3 mL). The organic layer was then washed with 5% aqueoussodium hydroxide solution (3×1.67 mL), saturated aqueous sodiumbicarbonate solution (3×1.67 mL) and brine (3×1.67 mL). The resultingsolution was dried over magnesium sulfate, filtered and concentrated invacuo to give a while solid (29.7 mg). The solid was dissolved in aminimum of hexanes-ethyl acetate (3:1) and combined with the crudeproduct from a subsequent identical reaction (364.7 mg), then purifiedby flash column chromatography [hexanes-ethyl acetate (3:1)] to give 27(332.8 mg, 1.27 mmol, 61%) as colorless oil above a white amorphoussolid. Note, additional impure material (33.9 mg) was recovered andsaved for purification at a later date, giving 65% yield overall. ¹H NMR(CDCl₃) δ 9.55 (1H, s), 7.32-0.04 (5H, m), 3.93 (4H, s), 2.78 (2H, s),1.68-1.47 (8H, m).

8-Benzyl-8-ethynyl-1,4-dioxaspiro[4,5]decane (28). Under a nitrogenatmosphere, potassium carbonate (343 mg, 2.48 mmol, 2 eq.) followed by asolution of 27 (324 mg, 1.24 mmol) in dry methanol (10 mL) were added toa stirred solution of freshly prepareddimethyl-1-diazo-2-oxopropylphosphonate (397 mg, 2.06 mmol, 1.5 eq.)(Müller, et. al) in dry methanol (10 mL) cooled in an ice bath. Thereaction mixture was stirred in the ice bath for 30 minutes, and thenstirred at room temperature overnight. The reaction was quenched withsaturated aqueous ammonium chloride solution (28.6 mL) and extractedwith methylene chloride (4×57 mL). The organic layers were combined anddried over magnesium sulfate, filtered, and dried in vacuo to give abeige solid (351.3 mg). The crude product was combined with the productfrom a previous identical reaction (36.3 mg) and dissolved inhexanes-ethyl acetate (5:1). The material was found to be only slightlysoluble, with thin layer chromatography showing the insoluble crystals(86.9 mg) to be almost pure product. The crystals were thereforeseparated by decanting, and the soluble material was purified by flashcolumn chromatography [hexanes-ethyl acetate (5:1)] to give a whitecrystalline solid (212.6 mg). The two crystalline samples were combinedto give pure product 28 (299.5 mg, 1.17 mmol, 85%): ¹H NMR (CDCl₃) δ7.32-7.21 (5H, m), 3.94 (4H, dd, J=2.5, 3 Hz), 2.76 (2H, s), 2.20 (1H,s), 2.01-1.91, 1.76-1.56 (8H, m); ¹³C NMR (CDCl₃) δ 137.6, 130.8, 127.9,126.7, 110.0, 108.9, 87.9, 72.3, 64.5, 64.4, 48.1, 37.0, 35.1, 31.8. MS(ES+) m/z 257.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₇H₂₀O₂+H 257.1542. found257.1541.

((8-Benzyl-1,4-dioxaspiro[4,5]decan-8-yl)ethynyl)trimethylsilane (29).Under nitrogen atmosphere and with stirring at 0° C., methyllithium (1.6M in hexanes, 4.78 mL, 7.65 mmol, 9.2 eq) was added dropwise to asolution of 28 (212.6 mg, 0.829 mmol) in dry THF (9.56 mL). The ice bathwas then removed and the mixture was stirred at room temperature for 25minutes. To the mixture was added dropwise a solution of trimethylsilylchloride (3.24 mL, 24.87 mmol, 30 eq) in dry THF (3.9 mL) and theresulting mixture was stirred at room temperature for 45 minutes. Duringthis time the solution became opaque and pale yellow. The reaction wasthen quenched with water (15 mL) and the aqueous layer was extractedwith methylene chloride/ethyl ether (1:2, 3×15 mL). The organic layerswere combined and washed with saturated aqueous sodium bicarbonatesolution (1×15 mL) and brine (1×15 mL), dried over magnesium sulfate,filtered, and concentrated in vacuo to give 29 (243.1 mg, 0.74 mmol,89%) as pale yellow crystals. The product was used in the next reactionwithout further purification: ¹H NMR (CDCl₃) δ 7.32-7.30 (5H, m), 3.95(4H, dd, J=2.5, 3.5 Hz), 2.72 (2H, s), 2.00-1.90, 1.71-1.50 (8H, m),0.14 (9H, s); ¹³C NMR (CDCl₃) δ 137.8, 130.9, 127.7, 126.5, 110.6,109.1, 88.2, 64.5, 64.4, 48.2, 38.0, 35.2, 31.9, 0.36. MS (EI) m/z 328.1[M⁺], 284.2, 237.2, 193.1, 99.1 (100%), 73.1, 59.0; HRMS (EI) calcd forC₂₀H₂₈O₂Si 328.1859. found 328.1856.

4-Benzyl-4-((trimethylsilyl)ethynyl)cyclohexanone (30). A sample of 29(243.1 mg, 0.74 mmol) was dissolved in acetone (2.89 mL) to give atranslucent, yellow solution. To the solution was added a solution of10% aqueous hydrochloric acid solution (1.15 mL) and the reactionmixture was stirred at room temperature for 5 hours. The mixture wasthen diluted with brine (25 mL) and the aqueous layer was extracted withmethylene chloride/ethyl ether (1:2, 3×20 mL). The organic layers werecombined and washed with saturated aqueous sodium bicarbonate solution(2×20 mL) and brine (1×20 mL), then dried over magnesium sulfate,filtered, and concentrated in vacuo to give the crude 30 as pale yellowcrystals (195 mg). The crystals were combined with the crude product ofa previous reaction (62.5 mg) and were purified by flash columnchromatography [hexanes-ethyl acetate (5:1)] to give 30 as whitecrystals (197.1 mg, 0.69 mmol, 74%). Note, an impure fraction wascollected and saved for future purification. ¹H NMR (CDCl₃) δ 7.31-7.24(5H, m), 2.81 (2H, s), 2.74 (2H, td, J=6, 14.5 Hz), 2.30 (2H, dt, J=2,15 Hz), 2.00 (2H, dt, J=3, 14 Hz), 1.68 (2H, dt, J=4, 13.5 Hz), 0.17(9H, s); ¹³C NMR (CDCl₃) δ 211.7, 137.1, 130.8, 128.0, 126.9, 109.0,47.6, 38.6, 38.0, 37.2, 0.29. MS (ESI+) m/z 285.1 [M+H]⁺, HRMS (ESI+)calcd for C₁₈H₂₄OSi+H 285.1675. found 285.1661.

4-Benzyl-4-((trimethylsilanyl)ethynyl)cyclohex-2-enone (31). Under anitrogen atmosphere and with stirring, a sample of 30 (50 mg, 0.176mmol) was suspended in dry THF (1.4 mL) and the solution was cooled to−78° C. in a dry ice/isopropanol bath. To the stirred mixture was addedlithium diisopropylamide (LDA, 2 M in THF/n-heptane/ethylbenzene, 132μL, 0.26 mmol) dropwise. The isopropanol bath was then removed and themixture was allowed to reach room temperature over 20 minutes. Themixture was again cooled to −78° C. and to it was added dropwise asolution of phenylselenenyl chloride (66.5 mg, 0.347 mmol, 2 eq) in dryTHF (0.42 mL). The isopropanol bath was removed and the reaction wasstirred at room temperature for 2 hours. During this time a yellowprecipitate appeared, indicating the formation of solid lithiumchloride. The reaction was then quenched with brine (5 mL) and theaqueous layer was extracted with dichloromethane/ethyl ether (1:2, 3×4mL). The organic layers were combined, washed with brine (1×4 mL), driedover magnesium sulfate, filtered, and concentrated in vacuo to give adark yellow-brown residue. This residue was dissolved in ethyl ether(2.82 mL) and hydrogen peroxide (30% aqueous, 3×42.3 μL) was added in 3successive portions, allowing the mixture to stir 5 minutes between eachaddition. After the third portion was added, the color of the mixturechanged from brown to pale yellow and translucent. The reaction mixturewas then washed with water (1×4 mL), saturated aqueous sodiumbicarbonate solution (2×4 mL), and brine (1×4 mL). The organic layerswere combined and dried over magnesium sulfate, filtered, andconcentrated in vacuo to give the crude product as a yellow oil (43.1mg). The crude was purified by flash column chromatography[hexanes-ethyl acetate (5:1)] to give 31 (19.2 mg, 0.068 mmol, 39%) as aclear oil: ¹H NMR (CDCl₃) δ 7.34-7.24 (5H, m), 6.74 (1H, dd, J=1.5, 10Hz), 5.94 (1H, dd, J=0.6, 10 Hz), 2.98 (1H, d, J=13 Hz), 2.89 (1H, d,J=13 Hz), 2.46 (2H, dt, J=5, 17 Hz), 2.08 (2H, td, J=1.5, 6 Hz), 0.16(9H, s); ¹³C NMR (CDCl₃) δ 209.1, 199.1, 152.1, 135.9, 130.9, 128.2,128.1, 127.3, 109.0, 106.1, 89.4, 46.4, 38.5, 35.0, 34.5, 0.11; MS(ESI+) m/z 283.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₈H₂₂OSi+H 283.1518.found 283.1508.

5-Benzyl-5-((trimethylsilyl)ethynyl)-4,5-dihydrobenzo[d]isoxazole (32).Under an argon atmosphere, ethyl formate (110.6 μL, 1.33 mmol, 5 eq)then sodium methoxide (71.7 mg, 1.33 mmol, 3 eq) were successively addedto a solution of 31 (75 mg, 0.265 mmol) in dry benzene (3.07 mL). Thismixture was stirred at room temperature for 1 hour, during which time acolor change from orange to red-brown occurred. The mixture was thendiluted with a mixture of methylene chloride/ethyl ether (1:2, 20 mL)and washed with saturated aqueous ammonium chloride solution (2×8 mL).The aqueous washings were combined and extracted with methylenechloride/ethyl ether (1:2, 3×10 mL). The organic layers were thencombined and washed with brine (1×10 mL), dried over magnesium sulfate,filtered, and concentrated in vacuo to give4-benzyl-6-hydroxymethylene-4-((trimethyl-silyl)ethynyl)cyclohex-2-enone(83 mg, 100%) as a yellow-brown oil. The product was used in the nextreaction without further purification: ¹H NMR (CDCl₃) δ 13.67 (1H, bs),7.45 (1H, bs), 7.23-7.16 (5H, m), 6.51 (1H, dd, J=1, 10.5 Hz), 5.98 (1H,d, J=10 Hz), 2.84 (1H, d, J=13 Hz), 2.68 (1H, d, J=13 Hz), 2.57 (1H, dd,J=1, 14.5 Hz), 2.40 (1H, d, J=15 Hz); ¹³C NMR (CDCl₃) δ 187.5, 168.7,151.4, 136.0, 130.8, 128.5, 128.1, 127.2, 127.1, 109.9, 108.0, 44.4,38.9, 34.6, 0.10.

Under a nitrogen atmosphere a solution of4-benzyl-6-hydroxymethylene-4-((trimethylsilyl)ethynyl)cyclohex-2-enone(76 mg, 0.245 mmol) in ethanol (6.13 mL) was added to a stirred solutionof hydroxylamine hydrochloride (177.6 mg, 2.53 mmol, 10.3 eq) in water(256 μL) at room temperature. The mixture was heated to 120° C. andstirred at reflux for 1 hour. After heating, the reaction mixture wasdiluted with water (10 mL) and the aqueous layer was extracted withdichloromethane/ethyl ether (1:2, 3×10 mL). The organic layers were thencombined and washed with saturated aqueous sodium bicarbonate solution(1×10 mL) and brine (2×10 mL), dried over magnesium sulfate, filtered,and concentrated in vacuo to give the crude product as a yellow-brownoil (76.4 mg). The crude was purified first by dissolving the product inpetroleum ether/ethyl ether (5:1) and decanting the soluble portion fromthe insoluble solid. The insoluble material was washed with petroleumether/ethyl ether (5:1) several times and the resulting mother liquorwas found by thin layer chromatography (TLC) to contain the desiredcompound. The mother liquor was purified by flash column chromatography[petroleum ether-ethyl ether (5:1)] to give 32 (17.1 mg, 0.055 mmol,22%) as a colorless oil: ¹H NMR (CDCl₃) δ 8.09 (1H, s), 7.32-7.22 (5H,m), 6.56 (1H, dd, J=1, 10 Hz), 6.0 (1H, d, J=10 Hz), 2.96 (1H, d? J=16Hz), 2.94 (1H, d, J=13 Hz), 2.83 (1H, d, J=16 Hz), 2.73 (1H, d, J=13Hz), 0.16 (9H, s); ¹³C NMR (CDCl₃) δ 164.4, 148.7, 139.3, 135.0, 131.0,128.0, 127.1, 114.4, 109.1, 88.4, 44.2, 39.2, 30.3, 0.13. MS (ESI+) m/z308.1 [M+H]⁺, HRMS (ESI+) calcd for C₁₉H₂₁NOSi+H 308.1471. found308.1467.

3-Benzyl-3-ethynyl-6-oxocyclohexa-1,4-dienecarbonitrile (MCE-13). Underan argon atmosphere and with stirring, a solution of 32 (17.1 mg, 0.055mmol) in anhydrous ethyl ether (1.27 mL) was added to a solution ofsodium methoxide (44.7 mg, 0.829 mmol, 15 eq) in dry methanol (1.03 mL).The mixture was stirred at room temperature for 2 hours. The reactionmixture was then diluted with methylene chloride/ethyl ether (1:2, 10mL) and washed with 5% aqueous hydrochloric acid solution (2×5 mL). Theaqueous layers were combined and extracted with methylene chloride/ethylether (1:2, 3×8 mL). The organic layers were then combined and washedwith water (2×8 mL) and brine (1×8 mL), dried over magnesium sulfate,filtered, and concentrated in vacuo to give a yellow oil (12.2 mg).

The following reaction step was calculated assuming the last reactiongave a quantitative yield. DDQ (11.76 mg, 0.0518 mmol, 1 eq) wasintroduced into a flask containing dhMCE-13 (12.2 mg, 0.0518 mmol, 1eq), and the contents of the flask were dissolved under a nitrogenatmosphere in dry benzene (1.5 mL). The mixture was heated under refluxand stirred for 20 minutes. The reaction mixture was then allowed tocool to room temperature and was filtered through a Pasteur pipette withcotton and rinsed with benzene. The resulting solution was concentratedin vacuo to give a crude product dark orange solid. The crude waspurified by flash column chromatography [hexanes-ethyl acetate (2:1)] togive MCE-13 (6.5 mg, 0.0277 mmol, 54%) as a white solid: ¹H NMR (CDCl₃)δ 7.50 (1H, d, J=3 Hz), 7.34-7.33 (3H, m), 7.20-7.18 (2H, m), 6.92 (1H,dd, J=3, 10 Hz), 6.34 (1H, d, J=10 Hz), 3.32 (1H, d, J=13 Hz), 3.20 (1H,d, J=13 Hz), 2.53 (1H, s).

Example 6 Synthesis and Characterization of MCE-6 and MCE-7

MCE-6 and MCE-7 were synthesized as described below and summarized inScheme 6.

Protocol for the synthesis of 34: To a stirred solution of 33 (1.5 mmol,472 mg) in dry THF (14 mL) was added dropwise at 0° C. under N₂ MeLi(1.6M in hexanes, 4 eq, 6 mmol, 3.75 mL). The yellow solution wasstirred at room temperature for 30 min. Then TMSC1 (98%, 4 eq, 6 mmol,785 μL) was added dropwise and the mixture was stirred at roomtemperature for 1 hour. After addition of 25 mL of H₂O, the two layerswere separated and the aqueous layer was extracted with Et₂O/CH₂Cl₂: 2/1(3×20 mL). The combined organic layers were washed with a saturatedNaHCO₃ aqueous solution (1×25 mL) and with brine (1×25 mL), dried overMgSO₄ and filtered. The filtrate was concentrated under reduced pressureto afford 630 mg of crude as a yellow oil. The crude was purified byflash column chromatography (Hexanes/EtOAc:10/1) to afford 34 (518 mg,89%) as a white solid. ¹H NMR (CDCl₃) δ 5.43-5.46 (m, 1H), 3.84-4.01 (m,4H), 1.34 (s, 3H), 1.01 (s, 3H), 0.86 (s, 3H), 0.13 (s, 9H); ¹³C NMR(CDCl₃) δ 148.3, 118.8, 115.8, 113.4, 84.7, 65.1, 65.0, 52.1, 43.1,42.6, 40.9, 40.4, 36.3, 35.5, 27.3, 26.3, 23.3, 23.2, 20.3, 19.7, 19.3,0.3.

Protocol for the synthesis of 35: To a stirred solution of 34 (0.914mmol, 353 mg) in dry CH₂Cl₂ (4.6 mL) were successively added underN₂K₂CO₃ (0.5 eq, 0.457 mmol, 63 mg) and Rh₂(cap)₄ (0.5%, 0.00455 mmol, 3mg). To the blue suspension was added t-BuOOH in once (5M in decane, 5eq, 4.57 mmol, 910 μL) so that the mixture turned immediately in redpurple. After 1 h 15 m of stirring at room temperature, Rh₂(cap)₄ (0.5%,0.00455 mmol, 3 mg) then t-BuOOH in once (5M in decane, 5 eq, 4.57 mmol,910 μL) were still added. The mixture was stirring at room temperaturefor an additional hour. The mixture was filtered through a pad of silicawith CH₂Cl₂/MeOH: 95/5 to get rid of the catalyst. The filtrate wasconcentrated under reduced pressure to give 682 mg of crude as a purpleresidue. The crude was purified by flash chromatography(Hexanes/EtOAc:5/1) to afford 35 (175 mg, 47%) as a white solid. ¹H NMR(CDCl₃) δ 5.86 (s, 1H), 3.87-4.38 (m, 4H), 2.77-2.85 (ddd, 1H, J=4.5,15.0, 17 Hz), 2.35-2.40 (dt, 1H, J=2.7, 17 Hz), 2.17-2.21 (dt, 1H,J=3.0, 12.8 Hz), 2.05-2.10 (ddd, 1H, J=2.6, 4.4, 12.8 Hz), 1.80-1.96 (m,3H), 1.61-1.75 (m, 4H), 1.43-1.50 (m, 2H), 1.40 (s, 3H), 1.04 (s, 3H),0.89 (s, 3H), 0.13 (s, 9H); ¹³C NMR (CDCl₃) δ 201.1, 172.7, 122.4,112.6, 109.1, 87.2, 65.2, 65.1, 50.7, 42.9, 42.5, 41.7, 39.3, 36.9,34.9, 34.6, 27.0, 23.3, 23.1, 20.5, 19.3, 0.1.

Protocol for the synthesis of 36: A stirred solution of 35 (0.307 mmol,123 mg) in dry THF (1.5 mL) was cooled to −78° C. and LDA (2M inTHF/heptane, 1.4 eq, 0.43 mmol, 215 μL) was added. The brown mixture wasstirred at room temperature for 20 min and then cooled back to −78° C. Acloudy solution of p-TsCN (95%, 1.7 eq, 0.522 mmol, 100 mg) in dry THF(1 mL) was added and the mixture was stirred at −78° C. for 45 min. Asaturated NH₄OH aqueous solution (1.5 mL) was then added and the mixturewas let to reach room temperature. It was extracted with EtOAc (3×10mL). The combined organic layers were washed with a saturated NaHCO₃aqueous solution (1×10 mL) and with brine (1×10 mL), dried over MgSO₄and filtered. The filtrate was concentrated under reduced pressure togive 158 mg of a crude product as a brown oil. This reaction wasconsidered to be complete and the crude was directly dissolved under N₂in dry benzene (9 mL). DDQ (98%, 1.5 eq, 0.460 mmol, 106 mg) was addedand the brown suspension was refluxed at 100° C. for 10 min. Aftercooling to room temperature, the suspension was filtered and thefiltrate was concentrated under reduced pressure to give 230 mg of crudeas a brown oil. The crude was purified by flash column chromatography(CH₂Cl₂/Acetone: 100/1) to afford 36 (75 mg, 57%) as a pale pink solid.¹H NMR (CDCl₃) δ 7.34 (s, 1H), 6.24 (s, 1H), 3.86-4.01 (m, 4H),2.78-2.31 (dt, 1H, J=3.0, 12.5 Hz), 2.01-2.09 (m, 1H), 1.93-1.99 (m,1H), 1.80-1.85 (m, 2H), 1.65-1.71 (m, 2H), 1.48-1.53 (m, 1H), 1.48 (s,3H), 1.38-1.41 (dd, 1H, J=2.0, 12.5 Hz), 1.06 (s, 3H), 0.88 (s, 3H),0.16 (s, 9H); ¹³C NMR (CDCl₃) δ 180.4, 168.9, 160.7, 121.5, 113.9,112.2, 109.9, 101.2, 92.4, 65.2, 65.1, 51.6, 43.2, 42.3, 40.7, 40.1,34.6, 27.0, 23.3, 21.5, 20.2, 18.9, −0.3.

Protocol for the synthesis of 37 (MCE-6): A suspension of 36 0.177 mmol,75 mg) in methanol (3.5 mL) was refluxed to get a total dissolution.Then, a 10% HCl aqueous solution (2 mL) was added to the hot solutionand the mixture was stirred at room temperature for 15 min. Afterneutralization at neutral pH with Et₃N to get neutral pH, the mixturewas concentrated in vacuo. The white residue was dissolved in H₂O (5 mL)and extracted with EtOAc (3×10 mL). The combined organic layers werewashed with a saturated NaHCO₃ aqueous solution (1×10 mL) and with brine(1×10 mL), dried over MgSO₄ and filtered. The filtrate was concentratedunder reduced pressure to give 78 mg of crude as a pale brown solid. Thecrude was purified by flash column chromatography (CH₂Cl₂/Acetone:100/1) to afford 37 (58 mg, 86%) as a white solid. ¹H NMR (CDCl₃) δ 7.37(s, 1H), 6.24 (s, 1H), 2.75-2.82 (ddd, 1H, J=6.7, 12.7, 16.5 Hz),2.51-2.56 (ddd, 1H, J=2.7, 5.7, 16.5 Hz), 2.33-2.37 (dt, 1H, J=3.1, 12.8Hz), 2.18-2.27 (m, 1H), 2.08-2.13 (ddd, 1H, J=2.7, 6.7, 12.8 Hz),1.81-1.91 (m, 2H), 1.65 (s, 3H), 1.50-1.55 (m, 1H), 1.43-1.46 (dd, 1H,J=2.7, 12.2 Hz), 1.18 (s, 3H), 1.13 (s, 3H), 0.10 (s, 9H); ¹³C NMR(CDCl₃) δ 214.2, 180.0, 167.1, 160.4, 122.3, 114.2, 113.7, 100.7, 93.1,53.8, 48.1, 41.9, 40.7, 39.4, 36.3, 34.2, 26.2, 22.1, 20.7, 20.0, −0.3.

Protocol for the synthesis of MCE-7: A solution of TBAF (98%, 3 eq,0.336 mmol, 90 mg) in THF (850 μL) was added to 37. The yellow solutionwas stirred at room temperature for 15 min. After dilution in EtOAc (8mL), the organic layer was washed with a saturated NaHCO₃ aqueoussolution (2×4 mL). The basic aqueous layers were extracted with EtOAc(2×4 mL). The combined organic layers were washed with brine (1×7 mL),dried over MgSO₄ and filtered. The filtrate was concentrated underreduced pressure to get 48 mg of crude as a pale yellow solid. The crudewas purified by flash column chromatography (CH₂Cl₂/Acetone: 99/1) toafford MCE-7 (28 mg, 81%) as a white solid. ¹H NMR (CDCl₃) δ 7.40 (s,1H), 6.27 (s, 1H), 2.77-2.84 (ddd, 1H, J 6.7, 12.7, 16.5 Hz), 2.53 (s,1H), 2.51-2.57 (ddd, 1H, J=2.7, 6.0, 19.2 Hz), 2.38-2.42 (dt, 1H, J=3.1,12.8 Hz), 2.23-2.29 (m, 1H), 2.10-2.14 (ddd, 1H, J=2.7, 6.7, 12.8 Hz),1.84-1.92 (m, 1H), 1.66 (s, 3H), 1.53-1.59 (m, 1H), 1.44-1.47 (dd, 1H,J=2.5, 7.5 Hz), 1.18 (s, 3H), 1.14 (s, 3H); ¹³C NMR (CDCl₃) δ 214.1,179.8, 166.7, 160.2, 122.5, 114.6, 113.4, 80.4, 75.6, 53.8, 48.1, 41.9,39.5, 39.4, 36.2, 34.2, 26.2, 22.2, 20.7, 20.0.

Overall yield of MCE-7 (from 33): 16%.

Example 7 Synthesis and Characterization of MCE-12

MCE-12 was synthesized as described below and summarized in Scheme 7.

Protocol for the synthesis of 34: To a stirred solution of 33 (2 mmol,629 mg) in dry THF (18 mL) was added dropwise at 0° C. under N₂ MeLi(1.6M in hexanes, 3 eq, 6 mmol, 3.75 mL). The yellow solution wasstirred at room temperature for 30 min. Then TMSC1 (98%, 3 eq, 6 mmol,780 μL) was added dropwise and the mixture was stirred at roomtemperature for 1 hour. After addition of 20 mL of H₂O, the two layerswere separated and the aqueous layer was extracted with Et₂O/CH₂Cl₂: 2/1(3×25 mL). The combined organic layers were washed with a saturatedNaHCO₃ aqueous solution (1×25 mL) and with brine (1×25 mL), dried overMgSO₄ and filtered. The filtrate was concentrated under reduced pressureto afford 34 (738 mg, 95% crude) of crude as a yellow solid. The crudewas used for the next deketalization step without further purification.

Protocol for the synthesis of A3: A suspension of 34 (1.91 mmol, 738 mg)in methanol (40 mL) was heated at 60° C. to get a total dissolution.Then, a 10% HCl aqueous solution (8 mL) was added to the hot solutionand the mixture was stirred at room temperature for 30 min. Afterneutralization at neutral pH with Et₃N to get neutral pH, the mixturewas concentrated in vacuo. The white residue was dissolved in H₂O (20mL) and extracted with EtOAc (3×25 mL). The combined organic layers werewashed with a saturated NaHCO₃ aqueous solution (1×25 mL) and with brine(1×25 mL), dried over MgSO₄ and filtered. The filtrate was concentratedunder reduced pressure to give 683 mg of crude as a pale yellow solid.The crude was purified by flash column chromatography (Hexanes/EtOAc:10/1) to afford A3 (537 mg, 82%) as a white solid. ¹H NMR (CDCl₃) δ5.47-5.49 (dd, 1H, J=3.0, 5.0 Hz), 2.68-2.75 (ddd, 1H, J=6.7, 12.7, 15.9Hz), 2.39-2.44 (ddd, 1H, J=3.0, 5.7, 15.9 Hz), 1.84-2.14 (m, 7H),1.72-1.78 (td, 1H, J=6.0, 12.7 Hz), 1.54-1.66 (m, 2H), 1.47 (s, 3H),1.31-1.38 (m, 2H), 1.23-1.29 (td, 1H, J=3.5, 13.0 Hz), 1.11 (s, 3H),1.08 (s, 3H), 0.14 (s, 9H); ¹³C NMR (CDCl₃) δ 217.3, 147.2, 120.1,115.1, 85.3, 54.2, 47.9, 42.5, 40.7, 40.2, 37.4, 36.3, 34.9, 26.3, 26.1,22.7, 22.0, 20.7, 19.2, 0.3.

Synthesis of A4. To a solution of A3 (589 mg, 1.69 mmol) in i-propanol(20 mL) was added aluminum i-propoxide (2.89 g, 14.2 mmol). The mixturewas heated under reflux overnight. To the reaction mixture was added 5%aqueous HCl solution (25 mL). The aqueous mixture was extracted withmethylene chloride (25 mL×4). The extract was washed with saturatedaqueous sodium bicarbonate (35 mL×1) and brine (35 mL×1), dried overMgSO₄, and filtered. The filtrate was evaporated in vacuo to give acrystalline solid (570 mg). The solid was purified by flashchromatography [hexanes-ethyl acetate (10:1, 7:1, and 5:1)] to give A4as a crystalline solid (190 mg, 32%): ¹H NMR (CDCl₃) δ 5.48 (1H, t,J=3.6 Hz), 3.43 (1H, d, J=3.3 Hz), 1.32, 0.97, 0.92 (each 3H, s), 0.13(9H, s).

Synthesis of A5. To a solution of A4 (308 mg, 0.89 mmol) in drymethylene chloride (12 mL) was added 2-(trimethylsilyl)ethoxymethylchloride (SEMCl, 480 μL, 2.7 mmol). Subsequently,N,N-diisopropylethylamine (DIEA, 740 μL, 4.48 mmol) was slowly added.The mixture was stirred at room temperature overnight. The reactionmixture was diluted with methylene chloride (35 mL) and water (10 mL).The organic layer was separated and washed with brine (10 mL×1), driedover MgSO₄, and filtered. The filtrate was evaporated in vacuo to give acrystalline solid (733 mg). The solid was purified by flashchromatography [hexanes-ethyl acetate (10:1)] to give A5 as an oil (424mg, 100%): ¹H NMR (CDCl₃) δ 5.45 (1H, dd, J=2.5 and 4.5 Hz), 4.74 (1H,d, J=7.3 Hz), 4.64 (1H, d, J=7.3 Hz), 3.65 (2H, m), 3.27 (1H, s), 1.31,0.94, 0.92 (each 3H, s), 0.13, 0.02 (each 9H, s); ¹³C NMR (CDCl₃) δ148.6, 118.4, 94.0, 91.8, 84.5, 82.1, 65.2, 48.6, 43.1, 41.0, 40.6,38.3, 36.4, 32.0, 28.9, 26.3, 23.3, 22.7, 22.6, 19.4, 19.2, 18.3, 0.4,−1.2

Synthesis of A6. To a solution of A5 (424 mg, 0.89 mmol) in methylenechloride (9 mL) was added t-butyl hydroperoxide (70% in water, 1.28 mL,8.93 mmol), followed by chromium trioxide (115 mg, 1.15 mmol) in an icebath. The mixture was stirred at room temperature for 3 h. The reactionmixture was diluted with methylene chloride/ether (1:2, 30 mL) and water(5 mL). The mixture was washed with 5% aqueous NaOH solution (15 mL×1),5% aqueous HCl solution (15 mL×1), saturated aqueous sodium bicarbonatesolution (15 mL×1), and brine (15 mL×1), dried over MgSO₄, and filtered.The filtrate was evaporated in vacuo to give a brown residue (529 mg).The residue was purified by flash chromatography [hexanes-ethyl acetate(7:1)] to give A6 as an amorphous solid (236 mg, 54%): ¹H NMR (CDCl₃) δ5.87 (1H, s), 4.72 (1H, d, J=7.3 Hz), 4.63 (1H, d, J=7.3 Hz), 3.65 (2H,m), 3.29 (1H, s), 2.81, 2.37, 2.18, 2.07 (each 1H, m), 1.37, 0.98, 0.96(each 3H, s), 0.13, 0.02 (each 9H, s); ¹³C NMR (CDCl₃) δ 201.2, 173.2,122.2, 109.3, 94.0, 87.0, 81.2, 65.4, 47.2, 42.5, 41.9, 39.4, 38.6,37.0, 34.8, 31.2, 28.8, 23.2, 22.8, 22.3, 18.9, 18.3, 0.1, −1.2.

Synthesis of A7. To a solution of A6 (49 mg, 0.10 mmol) in dry THF (325μL) was added LDA (2M solution in THF/n-heptane/ethylbenzene, 60 μL,0.12 mmol) at −78° C. (in a dry ice/i-propanol bath). The reactionmixture was stirred at room temperature for 20 min. To the mixture wasslowly added a cloudy solution of p-TsCN (95%, 31.3 mg, 0.17 mmol) indry THF (490 μL) at −78° C. The mixture was stirred at −78° C. for 45min, and then saturated aqueous ammonia solution (490 μL) was slowlyadded. The mixture was allowed to reach room temperature, and additionalsaturated aqueous ammonia solution (2 mL) was added. The mixture wasextracted with ethyl acetate (6 mL×3). The extract was washed withsaturated aqueous sodium bicarbonate solution (8 mL×1) and brine (8mL×1), dried over MgSO₄, and filtered. The filtrate was evaporated invacuo to give a residue (51 mg).

The mixture of the residue and DDQ (28 mg, 0.122 mmol) in dry benzene(2.3 mL) was heated under reflux for 30 min. After removal of insolublematter, the filtrate was evaporated in vacuo to give a residual oil (76mg). The residue was purified by flash chromatography [methylenechloride-acetone (130:1)] to give A7 as a crystalline solid (20 mg,40%): ¹H NMR (CDCl₃) δ 7.33 (1H, s), 6.25 (1H, s), 4.71 (1H, d, J=7.3Hz), 4.61 (1H, d, J=7.3 Hz), 3.62 (2H, m), 3.30 (1H, brs), 1.46, 0.99,0.97 (each 3H, s), 0.16, 0.02 (each 9H, s).

Synthesis of A8. A suspension of A7 (38 mg, 0.074 mmol) in 48% aqueousHF solution/CH₃CN (1:10, 2.7 mL) was stirred at room temperature for 24h. The reaction mixture was diluted with methylene chloride/ether (1:2,10 mL), washed with water (5 mL×3), saturated aqueous sodium bicarbonatesolution (5 mL×1), and brine (5 mL×1), dried over MgSO₄, and filtered.The filtrate was evaporated in vacuo to give A8 as a crystalline solid(25 mg, 88%): ¹H NMR (CDCl₃) δ 7.33 (1H, s), 6.26 (1H, s), 3.48 (1H,brs), 2.30 (1H, m), 2.01 (4H, m), 1.45, 0.99, 0.98 (each 3H, s), 0.16(9H, s); ¹³C NMR (CDCl₃) δ 180.5, 169.3, 160.9, 121.4, 114.0, 109.9,101.4, 92.3, 75.4, 47.5, 42.6, 40.9, 40.3, 38.8, 30.7, 28.7, 25.6, 22.4,21.4, 18.8, −0.2. This material was used for the next step withoutfurther purification.

Synthesis of MCE-12. A mixture of A8 (25 mg, 0.065 mmol) andtetra-(n-butyl)ammonium fluoride (TBAF, 52 mg, 0.20 mmol) in THF (500nM) was stirred at room temperature for 30 min. The mixture was dilutedwith ethyl acetate (8 mL), and washed with saturated aqueous sodiumbicarbonate solution (4 mL×2). The aqueous basic washings were extractedwith ethyl acetate (4 mL×2). The extract and the original organicsolution were combined. The combined organic solution was washed withbrine (8 mL×1), dried over MgSO₄, and filtered. The filtrate wasevaporated in vacuo to give an amorphous solid (19 mg). The solid waspurified by flash chromatography [hexanes-ethyl acetate (1:1)] to giveMCE-12 as a crystalline solid (14 mg, 70%): ¹H NMR (CDCl₃) δ 7.36 (1H,s), 6.29 (1H, s), 3.49 (1H, t, J=2.5 Hz), 2.46 (1H, s), 2.35 (1H, m),2.08 (2H, m), 1.96 (1H, m), 1.78 (2H, m), 1.48, 1.00, 0.98 (each 3H, s);¹³C NMR (CDCl₃) δ 180.2, 168.8, 160.7, 121.5, 114.3, 113.7, 80.9, 75.4,75.1, 47.5, 42.6, 40.2, 39.7, 38.7, 30.7, 28.6, 25.6, 22.3, 21.4, 18.8.

Example 8 Synthesis and Characterization of MCE-8, MCE-9, MCE-10 andMCE-11

MCE-8-11 were synthesized as described below and summarized in Scheme 8.

Protocol for the synthesis of B4: To a stirred solution of A3 (1.57mmol, 537 mg) in dry MeOH (25 mL) was added NaBH₄ (98%, 2 eq, 3.14 mmol,121 mg). The mixture was refluxed for 15 min under N₂. After cooling toroom temperature, the mixture was concentrated in vacuo to get a whiteresidue. The residue was diluted in CH₂Cl₂ (35 mL) and wash with H₂O(2×15 mL). The combined organic layers were extracted with CH₂Cl₂ (2×10mL). The combined organic layers were washed with brine (1×15 mL), driedover MgSO₄ and filtered. The filtrate was concentrated under reducedpressure to give 560 mg of crude as a pale brown solid. The crude waspurified by flash column chromatography (Hexanes/EtOAc: 5/1) to affordB4 (420 mg, 77%) as a white amorphous solid. ¹H NMR (CDCl₃) δ 5.43-5.44(dd, 1H, J=2.5, 5.0 Hz), 3.17-3.24 (m, 1H), 1.30 (s, 3H), 0.99 (s, 3H),0.85 (s, 3H), 0.13 (s, 9H); ¹³C NMR (CDCl₃) δ 148.6, 118.7, 115.5, 84.8,79.0, 53.7, 43.2, 40.9, 40.6, 39.4, 36.9, 36.3, 28.5, 27.9, 26.3, 23.4,19.6, 19.2, 15.8, 0.3.

Protocol for the synthesis of B5: To a stirred solution of B4 (0.725mmol, 250 mg) in dry CH₂Cl₂ (10 mL) were successively added SEM-Cl (90%,3 eq, 2.175 mmol, 430 μL) and DIEA (5 eq, 3.625 mmol, 600 μL). Themixture was stirred under nitrogen at room temperature overnight. Itturned into slightly yellow. After diluting with CH₂Cl₂ (30 mL), theorganic phase was washed with H₂O (2×15 mL) and with brine (1×15 mL),dried over MgSO₄ and filtered. The filtrate was concentrated underreduced pressure to give 538 mg of crude as a yellow oil/solid mix. Thecrude was purified by flash column chromatography (Hexanes/EtOAc: 10/1)to afford B5 (384 mg) as a colorless oil. A SEM-Cl residue remindedafter purification. We considered the reaction was quantitative (344 mg,100%) for the next step. ¹H NMR (CDCl₃) δ 5.41-5.43 (1H, dd, J=3.0, 4.5Hz), 4.80 (d, 1H, J=7.0 HZ), 4.67 (D, 1H, J=7.0 HZ), 3.60-3.68 (M, 2H),3.07-3.12 (dd, 1H, J=4.2, 11.7 Hz), 1.30 (s, 3H), 0.95 (s, 3H), 0.87 (s,3H), 0.13 (s, 9H), 0.02 (s, 9H); ¹³C NMR (CDCl₃) δ 148.7, 118.6, 115.6,94.2, 84.7, 84.6, 65.2, 54.1, 43.3, 40.9, 40.5, 39.2, 36.8, 36.3, 28.5,26.3, 24.5, 23.4, 19.6, 19.3, 18.3, 16.7, 0.3, −1.2.

Protocol for the synthesis of B6: To a stirred solution of B5 (0.725mmol, 344 mg) in CH₂Cl₂ at 0° C. were successively added t-BuOOH (70% inwater, 5 eq, 3.625 mmol, 500 μL) and CrO₃ (0.7 eq, 0.508 mmol, 51 mg).The ice bath was removed and the red purple mixture stirred at roomtemperature for 1 hour. Then, t-BuOOH (70% in water, 5 eq, 3.625 mmol,500 μL) and CrO₃ (0.7 eq, 0.508 mmol, 51 mg) were successively added at0° C. and the mixture was stirred for an additional hour. After dilutingwith Et₂O/CH₂Cl₂: 2/1 (50 mL), the organic layer was successively washedwith 10% NaOH aqueous solution (1×20 mL), with a 5% HCl aqueous solution(1×20 mL), with a saturated NaHCO₃ aqueous solution (1×20 mL) and withbrine (1×20 mL), dried over MgSO₄ and filtered. The filtrate wasconcentrated under reduced pressure to give 410 mg of crude as a redpurple oil. The crude was purified by flash column chromatography(Hexanes/EtOAc:7/1) to afford B6 (191 mg, 54%) as a white solid. ¹H NMR(CDCl₃) δ 5.84 (s, 1H), 4.79 (d, 1H, J=7 Hz), 4.66 (d, 1H, J=7 Hz), 3.65(t, 2H, J=8.5 Hz), 3.08-3.12 (dd, 1H, J=4.0, 12.0 Hz), 2.78-2.85 (ddd,1H, J=4.7, 15.0, 17.1 Hz), 2.36-2.40 (dt, 1H, J=3.0, 17.0 Hz), 2.19-2.23(dt, 1H, J=3.0, 12.8 Hz), 2.07-2.11 (ddd, 1H, J=3.1, 4.6, 12.8 Hz),1.81-1.97 (m, 4H), 1.63-1.74 (m, 2H), 1.35-1.44 (m, 1H), 1.37 (s, 3H),1.25-1.28 (m, 1H), 0.98-1.03 (m, 1H), 0.99 (s, 3H), 0.86-0.96 (m, 2H),0.91 (s, 3H), 0.13 (s, 9H), 0.02 (s, 9H); ¹³C NMR (CDCl₃) δ 201.1,172.8, 122.4, 109.3, 94.3, 87.2, 83.9, 63.4, 52.9, 42.7, 41.8, 39.5,39.3, 36.9, 36.0, 34.9, 28.4, 24.2, 23.2, 19.2, 18.3, 16.9, 0.1, −1.2.

Protocol for the synthesis of MCE-8: A stirred solution of B6 (0.333mmol, 163 mg) in dry THF (1.4 mL) was cooled to −78° C. and LDA (2M inTHF/heptane, 1.4 eq, 0.466 mmol, 230 μL) was added. The brown mixturewas stirred at room temperature for 20 min and then cooled back to −78°C. A cloudy solution of p-TsCN (95%, 1.7 eq, 0.556 mmol, 108 mg) in dryTHF (1.3 mL) was added and the mixture was stirred at −78° C. for 45min. A saturated NH₄OH aqueous solution (1.6 mL) was then added and themixture was let to reach room temperature. It was extracted with EtOAc(3×10 mL). The combined organic layers were washed with a saturatedNaHCO₃ aqueous solution (1×8 mL) and with brine (1×8 mL), dried overMgSO₄ and filtered. The filtrate was concentrated under reduced pressureto give 198 mg of a crude product as an orange oil. This reaction wasconsidered to be complete and the crude was directly dissolved under N₂in dry benzene (10 mL). DDQ (98%, 1.0 eq, 0.460 mmol, 77 mg) was addedand the brown suspension was refluxed at 100° C. for 30 min. Aftercooling to room temperature, the suspension was filtered and thefiltrate was concentrated under reduced pressure to give 220 mg of crudeas a brown oil. The crude was purified by flash column chromatography(CH₂Cl₂/Acetone: 140/1) to afford MCE-8 (89 mg, 52%) as a pale pinksolid. ¹H NMR (CDCl₃) δ 7.34 (s, 1H), 6.21 (s, 1H), 4.79 (d, 1H, J=7.0Hz), 4.66 (d, 1H, J=7.0 Hz), 3.63-3.67 (m, 2H), 3.08-3.11 (dd, 1H,J=4.5, 11.5 Hz), 2.30-2.33 (dt, 1H, J=3.0, 12.5 Hz), 1.46 (s, 3H), 1.0(s, 3H), 0.94 (s, 3H), 0.16 (s, 9H), 0.02 (s, 9H); ¹³C NMR (CDCl₃) δ180.4, 169.1, 160.7, 121.4, 114.0, 113.9, 101.1, 94.3, 92.4, 83.6, 65.4,54.0, 42.3, 40.7, 40.2, 39.8, 36.0, 28.4, 24.1, 21.6, 18.9, 18.3, 16.7,−0.3, −1.2.

Overall yield of MCE-8 (from 33): 32%.

Protocol for the synthesis of MCE-9: A suspension of MCE-8 (0.09 mmol,46 mg) in HF (48% aqueous)/CH₃CN: 1/10 (3.15 mL) was stirred at roomtemperature overnight. The mixture was diluted with Et₂O/CH₂Cl₂: 2/1 (15mL) and washed with water (3×8 mL), with a saturated NaHCO₃ aqueoussolution (3×8 mL), dried over MgSO₄ and filtered. The filtrate wasconcentrated under reduced pressure to afford 37 mg of crude product asa white solid. The crude product was purified by flash columnchromatography (Hexanes/EtOAc: 1.5/1) to afford MCE-9 (29 mg, 85%) as awhite solid. ¹H NMR (CDCl₃) δ 7.35 (s, 1H), 6.21 (s, 1H), 3.21-3.23 (m,1H), 2.30-2.34 (dt, 1H, J=3.2, 12.5 Hz), 2.04-2.12 (m, 1H), 1.72-1.90(m, 4H), 1.65 (bs, 1H), 1.47-1.58 (m, 2H), 1.45 (s, 3H), 1.03 (s, 3H),0.91-0.94 (m, 1H), 0.93 (s, 3H), 0.16 (s, 9H); ¹³C NMR (CDCl₃) δ 180.4,167.0, 160.7, 121.4, 114.0, 113.9, 101.1, 92.5, 78.0, 53.6, 42.4, 40.7,40.2, 39.9, 36.1, 28.4, 27.3, 21.6, 18.9, 15.8, −0.3.

Overall yield of MCE-9 (from 33): 27%.

Protocol for the synthesis of MCE-10: A solution of TBAF (98%, 3 eq,0.150 mmol, 40 mg) in THF (380 μL) was added to MCE-9 (0.0497 mmol, 19mg) and the yellow mixture was stirred at room temperature for 15 min.The mixture was then diluted with EtOAc (8 mL) and washed with asaturated NaHCO₃ aqueous solution (2×4 mL). The combined aqueous layerswere combined and extracted with EtOAc (2×4 mL). The organic layers werecombined, washed with brine (1×8 mL), dried over MgSO₄ and filtered. Thefiltrate was concentrated under reduced pressure to give 19 mg of crudeproduct as yellow oil/solid residue. The crude product was purified byflash column chromatography (Hexanes/EtOAc: 1/1) followed by apreparative TLC (Hexanes/EtOAc:1/1) to afford MCE-10 (8 mg, 53%) aswhite crystals. ¹H NMR (CDCl₃) δ 7.38 (s, 1H), 6.24 (s, 1H), 2.22-2.25(dd, 1H, J=5.0, 11.0 Hz), 2.48 (s, 1H), 2.34-2.38 (dt, 1H, J=3.2, 12.7Hz), 2.05-2.15 (m, 1H), 1.73-1.93 (m, 4H), 1.60 (bs, 1H), 1.45-1.58 (m,2H), 1.48 (s, 3H), 1.03 (s, 3H), 0.93-0.96 (m, 1H), 0.93 (s, 3H); ¹³CNMR (CDCl₃) δ 180.1, 168.6, 160.6, 121.7, 114.4, 113.7, 80.7, 78.1,75.3, 53.6, 42.5, 40.2, 40.0, 39.6, 36.1, 28.5, 27.4, 21.7, 19.0, 15.9.

Overall yield of MCE-10 (from 33): 14%.

Protocol for the synthesis of MCE-11: A mixture of MCE-8 (27 mg, 0.053mmol) and TBAF (40 mg, 0.15 mmol, 2.9 equiv.) in THF (405 μL) wasstirred at room temperature for 45 min. The reaction mixture was dilutedwith EtOAc (8 mL) and washed with saturated aqueous sodium bicarbonatesolution (4 mL×4). The washes were extracted with EtOAc (4 mL×2). Theextract and the original organic solution were combined, and then washedwith brine (8 mL×1), dried over MgSO₄, and filtered. The filtrate wasevaporated in vacuo to give a yellow oil (24 mg). The oil was purifiedby flash chromatography [hexanes-ethyl acetate (4:1)] to give MCE-11 asan amorphous solid (12 mg, 51%): ¹H NMR (CDCl₃) δ 7.37 (s, 1H), 6.23 (s,1H), 4.80 (d, 1H, J=7.3 Hz), 4.56 (d, 1H, J=7.3 Hz), 3.65 (dd, 2H,J=7.0, 9.7 Hz), 0.10 (dd, 1H, J=4.5, 11.5 Hz), 2.47 (s, 1H), 2.35 (dt,1H, J=3.2, 12.7 Hz), 2.10 (m, 1H), 1.68-1.99 (m, 4H), 1.61 (bs, 1H),1.48 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H), 0.02 (s, 9H); ¹³C NMR (CDCl₃)δ 180.1, 168.7, 160.6, 121.6, 114.4, 113.7, 94.3, 83.6, 80.7, 75.2,65.4, 53.9, 42.3, 40.1, 40.0, 39.8, 39.6, 36.0, 28.4, 24.1, 21.7, 18.9,18.3, 16.7, −1.2.

Example 9 Proposed Synthesis of Compounds 38 and 39

Compound 38 may be synthesized as described below and summarized inScheme 9.

For example, compound 38 may be synthesized from the known compound 2(Phansavath et al., 1998) by the sequence shown in Scheme 9.Nucleophilic addition of enolate of 2 to homobenzyl bromide would give40. Reduction of 40 with LiAlH₄, followed by oxidation with CrO₃ wouldgive 42. Wittig reaction of 42 with (chloromethyl)triphenylphosphoniumchloride (Mella et al., 1988, which is incorporated herein byreference), followed by dehydrochlorination with MeLi and subsequenttreatment with chlorotrimethylsilane (TMSCl) (Corey et al., 1973, whichis incorporated herein by reference) would afford 43. The ketal of 43would be removed under acidic conditions to yield 44. Enone 45 would beprepared by addition of phenylselenyl group to lithium enolate of 44 andsubsequent oxidation/elimination with 30% aqueous hydrogen peroxide. Atarget compound 38 would be obtained by cyanation of 45 withp-toluenesulfonyl cyanide (p-TsCN), followed by DDQ oxidation andsubsequent removal of TMS group under acidic conditions.

Similarly compound 39 (shown below) may be synthesized using1-bromo-2,2-diphenylethane, which is prepared from commerciallyavailable 2,2-diphenylethanol (Ohno et al., 2005, which is incorporated)in place of homobenzyl bromide in Scheme 9 above.

Example 10 Proposed Synthesis of Compound 46

Compound 46 may be synthesized as described below and summarized inScheme 10.

Compound 46 having t-butyl group has been designed as a compound with atypical hydrophobic group. Compound 46 would be synthesized from 2 using1-bromo-3,3-dimethylbutane (Hsiao et al, 1988, which is incorporatedherein by reference) in place of homobenzyl bromide in Scheme 9 above.

Example 11 Proposed Synthesis of Compound 48

Compound 48 may be synthesized as described below and summarized inScheme 11.

Compound 48, having a dimethylene amino group, would be synthesized from2 by the sequence shown in Scheme 11. Compound 49 would be prepared bynucleophilic addition of the enolate of 2 to commercially availableBr(CH₂)₂NHBoc. Compound 50 would be obtained from 49 by the samesequence as for 43 from 40 (see Scheme 9). The ketal of 50 would beremoved under acidic conditions, and subsequent reprotection with Boc₂Owould give 51. Enone 52 would be prepared by addition of phenylselenylgroup to lithium enolate of 51 and subsequent oxidation/elimination with30% aqueous hydrogen peroxide. Compound 48 (hydrochloride salt) would beobtained by cyanation of 52 with p-TsCN, followed by DDQ oxidation andsubsequent treatment with HCl in methanol.

Example 12 Proposed Synthesis of Compound 53

Compound 53 may be synthesized as described below and summarized inScheme 12.

Compound 53, having a trimethylene amino group, would be synthesizedfrom 2 as shown in Scheme 12 above. Nucleophilic addition of the enolateof 2 to commercially available 3-bromopropionitrile (Shuman et al.,1995, which is incorporated herein by reference) would give 54.Reduction of 54, followed by protection with Boc₂O, would produce 55.The ketone 57 would be obtained via 56 from 55 by the same sequence asfor 51. A target amine hydrochloride 53 would be prepared from 57 by thesame sequence as for 48 from 51.

Example 13 Proposed Synthesis of Compounds 58 and 59

Compounds 58 and 59 may be synthesized as described below and summarizedin Scheme 13.

Alcohol 58 and acid 59 may be synthesized by the sequence as shown inScheme 13. Nucleophilic addition of the enolate of 2 to vinyl bromidewould give 60. Compound 61 would be obtained from 60 by the samesequence as for 43 from 40 (see Scheme 9). Hydroboration of 61, followedby protection of hydroxyl group, would produce 62. Removal of the ketalof 62, followed by addition of phenylselenyl group and subsequentoxidation/elimination with 30% aqueous hydrogen peroxide would giveenone 63. Compound 64 would be produced by cyanation of 63 with p-TsCN,followed by DDQ oxidation. Deprotection of 64 would afford the desiredalcohol 58. Oxidation of 58 would give acid 59 (for example, Jonesoxidation, Ag₂O oxidation of aldehyde obtained from 58, etc).

Example 14 Proposed Synthesis of Compound 66

Compound 66 may be synthesized as described below and summarized inScheme 14.

For example, compound 66 would be synthesized from 2 by the sequenceshown in Scheme 14. Known compound 65 is prepared by nucleophilicaddition of the enolate of 2 to propargyl bromide. Sonogashira couplingbetween 65 and 2-iodo-1-(trimethylsilylethoxymethoxy)imidazole (Paul etal. 2002, which is incorporated herein by reference) would give 67.Compound 68 would be obtained from 67 by the same sequence as for 43from 40 (see Scheme 9). Usual methods (removal of a ketal, insertion ofa double bond by addition of phenylselenyl group and oxidativeelimination, cyanation, and DDQ oxidation) would give 69. Removal of theSEM and TMS groups of 69 under trifluoroacetic acid (TFA) in THF wouldgive 66, whose hydrochloride salt is water-soluble.

Example 15 Proposed Synthesis of Compounds of Formula VI

Compounds of formula VI, (for example, R=—Ph. —CH₂Ph, and —N═CHCH₂Ph)may be synthesized as described below and summarized in Scheme 15.

A facile synthetic sequence to compounds of formula VI is shown inScheme 13. Starting material V would be protected by benzaldehyde(Clarke et al., 2004, which is incorporated herein by reference) to give70. Swern oxidation of 70 would yield aldehyde 71. The treatment of 71with the Bestmann-Ohira reagent (Pietruszka et al., 2006, which isincorporated herein by reference) would produce acetylene 72.Deprotection of 72, followed by Dess-Martin oxidation, would afford 73.Desired compound VI would be obtained by aldol condensation betweencyanoacetone (Sauers et al., 2003, which is incorporated herein byreference) and 73. Applicants note that they are unaware of any previousreports of a six membered ring by this method. Starting materials Vwould be easily obtained or prepared. For example, known compounds 75a(V:R=phenyl) and 75b (V:R=benzyl) are prepared by condensation betweenformaldehyde and 2-phenylethanal (74a) and 3-phenylpropanal (74b) underbasic conditions, respectively (Rockendorf et al., 2002). Also, compound77 (V: R=—N═CHCH₂Ph) would be synthesized by condensation between2-phenylethanal (74a) and commercially available 76.

Example 16 Proposed Syntheses of Further Monocyclic Cyanoenones

Further monocyclic cyanoenones and intermediates thereof may besynthesized as described below and summarized in Schemes 16-18.

In Scheme 16, compounds C16 and C17 may be synthesized from C3 asfollows. Grignard reaction of methylmagnesium bromide with C3 would givetent-alcohol C32. C16 would be obtained from C32 via enone C33 by thesame sequence as for MCE-15 from 19 (see Scheme 4). Dehydroxylation ofC16 would afford C17.

Compound C18 would be synthesized from C35 via C36 by the same sequenceas for MCE-15 from 19 (see Scheme 4). The precursor C35 would beobtained from 5 via C34 by Grignard reaction of phenylmagnesium bromide,followed by modified Barton's dehydroxylation method.

Compound C19 would be synthesized from C3 by the same sequence as forMCE-15 from 19 (see Scheme 4). Alkali hydrolysis of C19 would give C20.Compound C21 would be obtained by treatment of C20 with oxalyl chloride,followed by amidation with ammonia.

C22 would be synthesized from 5 as follows. Condensation of 5 withhydroxylamine would give oxime C38. Dehydration of oxime C38 wouldproduce nitrile C39. Protected amine C40 would be prepared by reductionof C39 with NaBH₄—CoCl₂ and subsequent work-up with acidic conditions,followed by protection with Boc₂O. Enone C41 would be obtained byremoval of the ketal of C40 and subsequent protection of amino groupwith Boc₂O, followed by addition of phenylselenyl group and subsequentoxidation/elimination with 30% aqueous hydrogen peroxide. Compound C22would be produced by cyanation with p-TsCN and subsequent DDQ oxidation,followed by deprotection under hydrochloric acid-MeOH conditions.

Compound C23 would be synthesized from C39 via C42 by the same sequenceas for MCE-15 from 19 (see Scheme 4).

Compound C24 would be obtained from C43 by the same sequence as forMCE-15 from 19 (see Scheme 4). The precursor C43 would be prepared byprotection of the hydroxyl group of C4 with t-butylchlorodimethyl-silane(TBSCl). Removal of the TBS group of C24 with TBAF would give C25.

Compound C26 would be synthesized from C46 by the same sequence as forMCE-15 from 19 (see Scheme 4). The precursor C46 would be prepared byGrignard reaction of methylmagnesium bromide with aldehyde 5, followedby protection with TBSCl.

Deprotection of C26 would give C27. Oxidation of C27 with chromiumtrioxide-pyridine would give C28.

Compounds C48-C53 would be synthesized from C2 according to the samesequence as shown in Scheme 17. For example, C48 would be synthesized innine steps from C56, which would be obtained by alkylation of thelithium ester enolate C2 with iodoethane (reduction, oxidation, Wittigreaction, dehydrochlorination and trapping with TMSC1, deketalization,insertion of double bond with PhSeCl and subsequentoxidation/elimination, cyanation with p-TsCN, DDQ oxidation, anddeprotection with TBAF).

Compounds C54 and C55 would be synthesized as follows. Cyanation of thelithium ester enolate C2 with PhOCN or p-TsCN would give C61. CompoundC62 would be obtained from C61 in 4 steps (reduction, oxidation, Wittigreaction, and dehydrochlorination). Alkaline hydrolysis of C62, followedby treatment with iodomethane would afford methyl ester C63. Enone C64would be produced by removal of the ketal of C63, followed by additionof phenylselenyl group and subsequent oxidation/elimination. CompoundC54 would be prepared by cyanation of C64 with p-TsCN and subsequent DDQoxidation, followed by deprotection. Alkaline hydrolysis of C54 wouldyield C55.

Compounds C65-C68 and C70 would be synthesized from 5 by the samesequence as shown in Scheme 18. For example, C65 would be synthesized asfollows. Wittig reaction of 5 with (chloromethyl)-triphenylphosphoniumchloride, followed by dehydrochlorination with MeLi and subsequenttreatment with aqueous NH₄Cl solution would give C71. Methylation of theacetylide C71 with iodomethane would afford C72. C65 would be obtainedin 4 steps from C72 (deketalization, insertion of double bond, cyanationwith p-TsCN, DDQ oxidation). C69 would be synthesized from C68 byalkaline hydrolysis.

Example 17 Proposed Synthesis of Compounds of Formula VII

Compounds of formula VII, (for example, R=—CH₂NH₂ and —CO₂H) may besynthesized as described below and summarized in Schemes 19 or 20.

Compound D55 (VII: R=—CH₂NH₂) would be synthesized from a known compoundD56 (Honda et al., 2005, which is incorporated herein by reference) bythe sequence shown in Scheme 19. Schmidt reaction on D56 would give D57along with the lactam (Finlay et al., 1997, which is incorporated hereinby reference). Hydrogenolysis of D57, followed by protection with Boc₂O,would produce D58. Compound D59 would be prepared by oxidation of D58,followed by a Wittig reaction and subsequent trapping of acetylide withTMSC1. Chromium-mediated allylic oxidation of D59 would produce D60. Thedesired compound D55 would be obtained by cyanation of D60, followed byDDQ oxidation and subsequent removal of Boc and TMS groups under acidicconditions.

Similarly, compound D61 (VII: R=—CO₂H) would be synthesized from D57 bythe sequence shown in Scheme 20. Hydrolysis of nitrile of D57 underacidic conditions would give acid D62. Hydrogenolysis of D62, followedby methylation, would yield methyl ester D63. Compound D65 would beprepared via D64 from D63 by the same sequence as for D60. The desiredcompound D61 would be obtained by cyanation of D65, followed by DDQoxidation and subsequent removal of methyl and TMS groups under basicconditions.

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A compound of the formula:

wherein: R₁, R₂, R₃, R₄, R₅ and R₆ are each independently: hydrogen,hydroxy, amino, cyano, or alkyl_((C≦18)), alkenyl_((C≦18)),alkynyl_((C≦18)); aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),aralkoxy_((C≦18)), heteroaryloxy_((C≦18)), heteroaralkoxy_((C≦18)),acyloxy_((C≦18)), alkyl-amino_((C≦18)), dialkylamino_((C≦18)),alkoxyamino_((C≦18)), alkenyl-amino_((C≦18)), alkynylamino_((C≦18)),arylamino_((C≦18)), aralkylamino_((C≦18)), heteroarylamino_((C≦18)),heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),amido_((C≦18)), alkylideneamino_((C≦18)); aralkylideneamino_((C≦18)); R₁and R₃ are taken together and are alkanediyl_((C≦18)),alkenediyl_((C≦18)), arenediyl_((C≦18)), alkoxydiyl_((C≦18)),alkenyloxydiyl_((C≦18)), alkylaminodiyl_((C≦18)),alkenylaminodiyl_((C≦18)), alkenylaminooxydiyl_((C≦18)),alkenyl-aminothiodiyl_((C≦18)), with R₂, R₄, R₅ and R₆ as defined above;or R₃ and R₅ are taken together and are alkanediyl_((C≦18)),alkenediyl_((C≦18)), arenediyl_((C≦18)), alkoxydiyl_((C≦18)),alkenyloxydiyl_((C≦18)), alkylaminodiyl_((C≦18)),alkenylaminodiyl_((C≦18)), alkenylaminooxydiyl_((C≦18)),alkenyl-aminothiodiyl_((C≦18)), with R₁, R₂, R₄ and R₆ as defined above;provided that: R₄ is absent when the atom to which it is bound formspart of a double bond; R₆ is absent when the atom to which it is boundforms part of a double bond; neither R₁ nor R₂ is hydrogen; and R₁ andR₂ are not both methyl; or a pharmaceutically acceptable salt ortautomer of the formula. 2-5. (canceled)
 6. The compound of claim 1further defined as:

wherein: R₂ and R₅ are each independently: hydrogen, hydroxy, amino,cyano, or alkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)),aryl_((C≦18)), aralkyl_((C≦18)), heteroaryl_((C≦18)),heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),aralkoxy_((C≦18)), heteroaryloxy_((C≦18)), heteroaralkoxy_((C≦18)),acyloxy_((C≦18)), alkyl-amino_((C≦18)), dialkylamino_((C≦18)),alkoxyamino_((C≦18)), alkenyl-amino_((C≦18)), alkynylamino_((C≦18)),arylamino_((C≦18)), aralkylamino_((C≦18)), heteroarylamino_((C≦18)),heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),amido_((C≦18)), alkylideneamino_((C≦8)), aralkylideneamino_((C≦18)),provided that R₂ is not hydrogen; and R₇ and R₈ are each independently:hydrogen, hydroxy, halo, oxo, amino, hydroxyamino, nitro, imino, cyano,azido, mercapto, or thio; or alkyl_((C≦6)), alkenyl_((C≦6)),alkynyl_((C≦6)), aryl_((C≦6)), aralkyl_((C≦6)), heteroaryl_((C≦6)),heteroaralkyl_((C≦6)), acyl_((C≦6)), alkylidene_((C≦6)), alkoxy_((C≦6)),alkenyloxy_((C≦6)), alkynyloxy_((C≦6)), aryloxy_((C≦6)),aralkoxy_((C≦6)), heteroaryloxy_((C≦6)), heteroaralkoxy_((C≦6)),acyloxy_((C≦6)), alkyl-amino_((C≦6)), dialkylamino_((C≦6)),alkoxyamino_((C≦6)), alkenylamino_((C≦6)), alkynylamino_((C≦6)),arylamino_((C≦6)), aralkylamino_((C≦6)), heteroarylamino_((C≦6)),heteroaralkylamino_((C≦6)), alkylsulfonyl-amino_((C≦6)), amido_((C≦6)),alkylimino_((C≦6)), alkenylimino_((C≦6)), alkynylimino_((C≦6)),arylimino_((C≦6)), aralkylimino_((C≦6)), hetero-arylimino_((C≦6)),heteroaralkylimino_((C≦6)), acylimino_((C≦6)), alkylthio_((C≦6)),alkenylthio_((C≦6)), alkynylthio_((C≦6)), arylthio_((C≦6)),aralkyl-thio_((C≦6)), heteroarylthio_((C≦6)), heteroaralkylthio_((C≦6)),acylthio_((C≦6)), thioacyl_((C≦6)), alkylsulfonyl_((C≦6)),alkenylsulfonyl_((C≦6)), alkynyl-sulfonyl_((C≦6)), arylsulfonyl_((C≦6)),aralkylsulfonyl_((C≦6)), heteroaryl-sulfonyl_((C≦6)),heteroaralkylsulfonyl_((C≦6)), alkylammonium_((C≦6)),alkylsulfonium_((C≦6)), alkylsilyl_((C≦6)), alkylsilyloxy_((C≦6)), or asubstituted version of any of these groups; or a pharmaceuticallyacceptable salt or tautomer of the formula. 7-8. (canceled)
 9. Thecompound of claim 1 further defined as:

wherein: R₁ and R₂ are independently: hydroxy, amino, cyano, oralkyl_((C≦18)), alkenyl_((C≦18)), alkynyl_((C≦18)), aryl_((C≦18)),aralkyl_((C≦18)), heteroaryl_((C≦18)), heteroaralkyl_((C≦18)),acyl_((c≦18)), alkoxy_((C≦18)), alkenyloxy_((C≦18)),alkynyloxy_((C≦18)), aryloxy_((C≦18)), aralkoxy_((C≦18)),heteroaryloxy_((C≦18)), heteroaralkoxy_((C≦18)), acyloxy_((C≦18)),alkyl-amino_((C≦18)), dialkylamino_((C≦18)), alkoxyamino_((C≦18)),alkenyl-amino_((C≦18)), alkynylamino_((C≦18)), arylamino_((C≦18)),aralkylamino_((C≦18)), heteroarylamino_((C≦18)),heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),amido_((C≦18)), alkylideneamino_((C≦18)), aralkylideneamino_((C≦18)), ora substituted version of any of these groups, provided that R₁ and R₂are not both methyl; or a pharmaceutically acceptable salt or tautomerof the formula.
 10. (canceled)
 11. The compound of claim 9, wherein thebond between atoms 4 and 5 is a double bond.
 12. The compound of claim11, wherein R₂ is alkenyl_((C≦12)), substituted alkenyl_((C≦12)),alkynyl_((C≦12)) or substituted alkynyl_((C≦12)).
 13. The compound ofclaim 12, wherein R₂ is alkynyl_((C≦8)) or substituted alkynyl_((C≦8)).14. The compound of claim 13, wherein R₂ is —C≡C—R₉, wherein R₉ is:hydrogen or cyano; or alkyl_((C≦6)), aryl_((C≦6)), acyl_((C≦6)),alkylsilyl_((C≦6)) or a substituted version of any of these groups. 15.The compound of claim 14, wherein R₉ is hydrogen.
 16. The compound ofclaim 14, wherein R₉ is —Si(CH₃)₂C(CH₃)₃. 17-19. (canceled)
 20. Thecompound of claim 12, wherein R₂ is alkenyl_((C≦8)) or substitutedalkenyl_((C≦8)).
 21. The compound of claim 20, wherein R₂ is ethenyl.22. The compound of claim 1, wherein R₂ is cyano. 23-29. (canceled) 30.The compound of claim 11, wherein R₁ is alkyl_((C≦8)) or substitutedalkyl_((C≦8)).
 31. The compound of claim 30, wherein R₁ is methyl orethyl. 32-36. (canceled)
 37. The compound of claim 11, wherein R₁ isaralkyl_((C≦18)), substituted aralkyl_((C≦18)), heteroaralkyl_((C≦18))or substituted heteroaralkyl_((C≦18)). 38-63. (canceled)
 63. Thecompound of claim 11, wherein R₁ is cyano.
 64. The compound according toclaim 1, further defined as:

wherein R₁ is: cyano, or alkynyl_((C≦18)), aryl_((C≦18)),aralkyl_((C≦18)), heteroaryl_((C≦18)), alkyl_((C≦18)), alkenyl_((C≦18)),heteroaralkyl_((C≦18)), acyl_((C≦18)), alkoxy_((C≦18)),alkenyloxy_((C≦18)), alkynyloxy_((C≦18)), aryloxy_((C≦18)),aralkoxy_((C≦18)), heteroaryloxy_((C≦18)), heteroaralkoxy_((C≦18)),acyloxy_((C≦18)), alkylamino_((C≦18)), dialkylamino_((C≦18)),alkoxyamino_((C≦18)), alkenylamino_((C≦18)), alkynylamino_((C≦18)),arylamino_((C≦18)), aralkylamino_((C≦18)), heteroarylamino_((C≦18)),heteroaralkylamino_((C≦18)), alkylsulfonylamino_((C≦18)),amido_((C≦18)), alkylideneamino_((C≦18)), aralkylideneamino_((C≦18)), ora substituted version of any of these groups; or a pharmaceuticallyacceptable salt or tautomer of the formula. 65-66. (canceled)
 67. Thecompound of claim 6, wherein R₅ is hydrogen. 68-70. (canceled)
 71. Acompound selected from the group consisting of:

72-76. (canceled)
 77. A pharmaceutical composition comprising as anactive ingredient a compound of claim 1 and a pharmaceuticallyacceptable carrier. 78-174. (canceled)